Late-pcr

ABSTRACT

A non-symmetric polymerase chain reaction (PCR) amplification method employing a limiting primer in low concentration whose concentration-adjusted melting point at least equals, and preferably exceeds, that of the excess primer, the latter in turn not being more than 25° C. below the melting temperature of the amplicon. Assays employing such amplification and labeled hybridization probes, including assays that include a detection step following primer extension or a low-temperature probe, or both. Kits for performing such assays and primer or primer-and-probe sets for performing the foregoing amplifications and assays.

TECHNICAL FIELD

[0001] This invention relates to amplification of nucleic acid sequencesby methods employing, in whole or part, exponential amplification by thepolymerase chain reaction (PCR) process.

BACKGROUND

[0002] The polymerase chain reaction (PCR) is widely used to amplifystretches of DNA, including cDNA reverse transcribed from RNA, forassays for diagnostic and other purposes. See U.S. Pat. Nos. 4,683,202,4,683,195 and 4,965,188. See, generally, PCR PROTOCOLS, a Guide toMethods and Applications, Innis et al. eds., Academic Press (San Diego,Calif. (USA) 1990). PCR reactions are generally designed to besymmetric, that is, to make double-stranded copies by utilizing aforward primer and a reverse primer in equimolar concentrations. The twoprimers are designed to have “melting temperatures,” or “T_(m)'s” thatare “balanced” (Innis et al., page 9), which is generally understood tomean equal or within a few degrees (° C.) of one another. A commonlyused computer software program for primer design warns users to avoidhigh T_(m) difference, and has an automatic T_(m) matching feature.(Oligo® Primer Analysis Software Manual, version 6.0 for Windows,Molecular Biology Insights, Inc., Sixth Edition, March 2000). TheT_(m)'s of linear primers comprised of deoxyribonucleotides (DNA) havebeen commonly determined by the “percent GC” method (Innis et al., page9) or the “2 (A+T) plus 4 (G+C)” method (Wallace et al. (1979)“Hybridization of Synthetic Oligodeoxyribonucletides to phi chi 174 DNA:the Effect of a Single Base Pair Mismatch,” Nucleic Acids Res. 6 (11):3543-3557) or the “Nearest Neighbor” method (SantaLucia, J. (1998) “AUnified view of Paymer, Dumbbell, and Oligonucleotide DNA NearestNeighbor Thermodynamics,” Proc. Natl. Acad. Sci. USA 95: 1460-1465;Allawi, H. T. and SantaLucia, J. (1997) “Thermodynamics and NMR ofInternal G·T Mismatches In DNA,” Biochem. 36: 10581-10594).

[0003] PCR is a repeated series of steps of denaturation, or strandmelting, to create single-stranded templates; primer annealing; andprimer extension by a thermally stable DNA polymerase such as Thermusaquaticus (Taq) DNA polymerase. A typical three-step PCR protocol (seeInnis et al., Chapter 1) may include denaturation, or strand melting, at93-95° C. for more than 5 sec, primer annealing at 55-65° C. for 10-60sec, and primer extension for 15-120 sec at a temperature at which thepolymerase is highly active, for example, 72° C. for Taq DNA polymerase.A typical two-step PCR protocol may differ by having the sametemperature for primer annealing as for primer extension, for example,60° C. or 72° C. For either three-step PCR or two-step PCR, anamplification involves cycling the reaction mixture through theforegoing series of steps numerous times, typically 25-40 times. Duringthe course of the reaction the times and temperatures of individualsteps in the reaction may remain unchanged from cycle to cycle, or theymay be changed at one or more points in the course of the reaction topromote efficiency or enhance selectivity. In addition to the pair ofprimers and target nucleic acid a PCR reaction mixture typicallycontains each of the four deoxyribonucleotide 5′triphosphates (dNTPs) atequimolar concentrations, a thermostable polymerase, a divalent cation,and a buffering agent. A reverse transcriptase is included for RNAtargets, unless the polymerase possesses that activity. The volume ofsuch reactions is typically 25-100 μl. Multiple target sequences can beamplified in the same reaction. In the case of cDNA amplification, PCRis preceded by a separate reaction for reverse transcription of RNA intocDNA, unless the polymerase used in the PCR possesses reversetranscriptase activity. The number of cycles for a particular PCRamplification depends on several factors including: a) the amount of thestarting material, b) the efficiency of the reaction, and c) the methodand sensitivity of detection or subsequent analysis of the product.Cycling conditions, reagent concentrations, primer design, andappropriate apparatuses for typical cyclic amplification reactions arewell known in the art (see, for example, Ausubel, F. Current Protocolsin Molecular Biology (1988) Chapter 15: “The Polymerase Chain Reaction,”J. Wiley (New York, N.Y. (USA)).

[0004] Ideally, each strand of each amplicon molecule binds a primer atone end and serves as a template for a subsequent round of synthesis.The rate of generation of primer extension products, or amplicons, isthus exponential, doubling during each cycle. The amplicons include bothplus (+) and minus (−) strands, which hybridize to one another to formdouble strands. To differentiate typical PCR from special variationsdescribed herein, we refer to typical PCR as “symmetric” PCR. SymmetricPCR thus results in an exponential increase of one or moredouble-stranded amplicon molecules, and both strands of each ampliconaccumulate in equal amounts during each round of replication. Theefficiency of exponential amplification via symmetric PCR eventuallydeclines, and the rate of amplicon accumulation slows down and stops.Kinetic analysis of symmetric PCR reveals that reactions are composedof: a) an undetected amplification phase (initial cycles) during whichboth strands of the target sequence increase exponentially, but theamount of the product thus far accumulated is below the detectable levelfor the particular method of detection in use; b) a detectedamplification phase (additional cycles) during which both strands of thetarget sequence continue to increase in parallel and the amount of theproduct is detectable; c) a plateau phase (terminal cycles) during whichsynthesis of both strands of the amplicon gradually stops and the amountof product no longer increases. Symmetric reactions slow down and stopbecause the increasing concentrations of complementary amplicon strandshybridize to each other (reanneal), and this out-competes the ability ofthe separate primers to hybridize to their respective target strands.Typically reactions are run long enough to guarantee accumulation of adetectable amount of product, without regard to the exact number ofcycles needed to accomplish that purpose.

[0005] Analysis of the amplified product is done by any of severalmeans. For instance, gel electrophoresis or, more recently, capillaryelectrophoresis has been widely used to separate amplified targetsequences, or “amplicons”, according to size. Bands on a gel aretypically made visible by use of an intercalating dye, such as ethidiumbromide or SYBR® Green, or by transferring the nucleic acid to amembrane and then visualizing it with a radioactively or fluorescentlylabeled hybridization probe. Analysis by sequencing most commonlyinvolves further amplification, using one primer in each of fourreaction vessels together with a different dideoxy dNTP. Under theseconditions each reaction generates a linear amplification productcomprised of a set of oligonucleotides ending in A, T, C or G dependingon which dideoxy dNTP was included in the reaction. See, for example,U.S. Pat. No. 5,075,216.

[0006] “Real-time” PCR refers to PCR reactions in which a reporter,typically a fluorescent moiety, is present to monitor the accumulationof the amplicon by a change in signal during the reaction. Such moietiesinclude an intercalating dye, such as SYBR® Green, or a hybridizationprobe (whether or not extendable as a primer). One real-time PCR method,the 5′ nuclease process, utilizes labeled linear probes, for exampledual fluorescent labeled probes (“TaqMan™ probes”), that are digested bythe DNA polymerase during the primer extension step, resulting in adetectable signal change (see U.S. Pat. Nos. 5,210,015, 5,487,972 and5,538,848). Another method utilizes a dye that fluoresces when incontact with double-stranded DNA (see U.S. Pat. No. 5,994,056). A thirdmethod utilizes dual fluorescent labeled probes such as “molecularbeacon probes”, which are hairpin probes having a fluorophore at one endand a quencher at the other end, and which open and fluoresce whenhybridized to their target sequence (see U.S. Pat. Nos. 5,925,517,6,103,476 and 6,365,729). Other fluorescent labeled probes useful forreal-time PCR include Scorpion primers, (primers that have a hairpinprobe sequence (containing a fluorophore and a quencher moieties locatedin close proximity on the hairpin stem) linked to their 5′ end via a PCRstopper such that fluorescence occurs only when the specific probesequence binds to its complement within the same strand of DNA afterextension of the primers during PCR; Whitcombe et al. (1999) “Detectionof PCR Products Using Self-Probing Amplicons and Fluorescence,” Nat.Biotechnol. 17: 804-807), Amplifluor primers (primers that have ahairpin probe sequence (containing a fluorophore and a quencher moietieslocated in close proximity on the hairpin stem) linked to their 5′ endsuch that fluorescence occurs only when the hairpin unfolds uponreplication of the primer following its incorporation into anamplification product; Nazarenko et al. (1997) “A Closed Tube Format forAmplification and Detection of DNA Based on Energy Transfer,” NucleicAcids Res. 15: 2516-21, Eclipse probes (linear DNA probes that have aminor-groove binding (MGB) protein-quencher complex positioned at the5′-end of the probe and a fluorophore located at the 3′-end of the probesuch that fluorescence only occurs when the probe anneals to a targetsequence aided by the MGB protein binding to NDA and the quencher movesaway from the fluorophore, (Afonina et al., (2002) “Minor GrooveBinder-Conjugated DNA Probes for Quantitative DNA Detection byHybridization-Triggered Fluorescence,” Biotechniques 32: 946-9), FRETprobes (a pair of random coil, or linear, probes, each of which isfluorescently labeled, that hybridize adjacently on a target sequence,causing their labels to interact by fluorescence resonance energytransfer (“FRET”) and produce a detectable signal change), anddouble-stranded fluorescent probes, (Li, Q. et al. (2002) “A New Classof Homogeneous Nucleic Acid Probes Based on Specific DisplacementHybridization,” Nucl. Acid Res. 30: (2)e5). Probes that are not to becut, hydrolyzed, or extended (that is, probes that are not primers) aretypically designed to disengage from their template either prior to orduring the primer extension step of PCR so they will not interfere withthis phase of the reaction. For probes such as molecular beacon probes,the melting temperature of the probe is generally 7-10° C. higher thanthe temperature used to anneal the primers. In practice this means thatthe melting temperature of the probe is higher than the meltingtemperature of the primer which hybridizes to the same strand as theprobe (Mackay, I. M. (2002) “Survey and Summary: Real-time PCR inVirology”, Nucleic Acids Res. 30(6): 1292-1305). Thus, as thetemperature of the reaction is cooled following strand-melting at 95° C.the probe hybridizes to its target strand (hereafter (+) strand)followed by hybridization of the primer for the (+) strand, as thereaction approaches the annealing temperature. As the reaction is warmedagain at the end of the annealing step the probe should fall off of the(+) strand while the primer extends along the (+) strand. Thus, theintent is that the probe should not interfere with primer extension.Hybridization and extension of the other primer on the complementary (−)strand also takes place during these steps. A second probe targeted tothe (−) strand may also be present in the reaction.

[0007] A technique that has found limited use for making single-strandedDNA directly in a PCR reaction is “asymmetric PCR.” Gyllensten andErlich, “Generation of Single-Stranded DNA by the polymerase chainreaction and its application to direct sequencing of the HLA-DQA Locus,”Proc. Natl. Acad. Sci. (USA) 85: 7652-7656 (1988); Gyllensten, U. B. andErlich, H. A. (1991) “Methods for generating single stranded DNA by thepolymerase chain reaction” U.S. Pat. No. 5,066,584, Nov. 19, 1991.Asymmetric PCR differs from symmetric PCR in that one of the primers isadded in limiting amount, typically {fraction (1/100)}^(th) to ⅕^(th) ofthe concentration of the other primer. Double-stranded ampliconaccumulates during the early temperature cycles, as in symmetric PCR,but one primer is depleted, typically after 15-25 PCR cycles, dependingon the number of starting templates. Linear amplification of one strandtakes place during subsequent cycles utilizing the undepleted primer.Primers used in asymmetric PCR reactions reported in the literature,including the Gyllensten patent, are often the same primers known foruse in symmetric PCR. Poddar (Poddar, S. (2000) “Symmetric vs.Asymmetric PCR and Molecular Beacon Probe in the Detection of a TargetGene of Adenovirus,” Mol. Cell Probes 14: 25-32 compared symmetric andasymmetric PCR for amplifying an adenovirus substrate by an end-pointassay that included 40 thermal cycles. He reported that a primers ratioof 50:1 was optimal and that asymmetric PCR assays had bettersensitivity that, however, dropped significantly for dilute substratesolutions that presumably contained lower numbers of target molecules.

[0008] Although asymmetric PCR has been known since 1988, it has notbeen extensively used as a technique because of the need to spend agreat deal of time optimizing the experimental conditions for eachamplicon. J. K. Ball and R. Curran (1997) “Production of Single-StrandedDNA Using a Uracil-N-glycosylase-Mediated Asymmetric Polymerase ChainReaction Method,” Analytical Biochemistry 253: 264-267, states: “Toensure that asymmetric amplification occurs several replicate tubescontaining different concentrations of each primer are set up, and forthis reason the technique is not used extensively.”

SUMMARY

[0009] As used herein, certain terms have defined meanings, as follows:

[0010] T_(m), or melting temperature, of an oligonucleotide describesthe temperature (in degrees Celsius) at which 50% of the molecules in apopulation of a single-stranded oligonucleotide are hybridized to theircomplementary sequence and 50% of the molecules in the population arenot-hybridized to said complementary sequence. The T_(m) of a primer orprobe can be determined empirically by means of a melting curve. In somecases it can also be calculated. For the design of symmetric andasymmetric PCR primer pairs, balanced T_(m)'s are generally calculatedby one of the three methods discussed earlier, that is, the “% GC”, orthe “2(A+T) plus 4 (G+C)”, or “Nearest Neighbor” formula at some chosenset of conditions of monovalent salt concentration and primerconcentration. In the case of Nearest Neighbor calculations the T_(m)'sof both primers will depend on the concentrations chosen for use incalculation or measurement, the difference between the T_(m)'s of thetwo primers will not change substantially as long as the primerconcentrations are equimolar, as they normally are with respect to PCRprimer measurements and calculations. T_(m[1]) describes the calculatedT_(m) of a PCR primer at particular standard conditions of 1 micromolar(1 μM 10⁻⁶M) primer concentration, and 0.07 molar monovalent cations. Inthis application, unless otherwise stated, T_(m[1]) is calculated usingNearest Neighbor formula, T_(m)=ΔH/(ΔS+R 1 n (C/2))−273.15+12 log [M].This formula is based on the published formula (Le Novere, N. (2001),“MELTING, Computing the Melting Temperature of Nucleic Acid Duplex,”Bioinformatics 17: 1226-7). ΔH is the enthalpy and ΔS is the entropy(both ΔH and ΔS calculations are based on Allawi and SantaLucia, 1997),C is the concentration of the oligonucleotide (10⁻⁶M), R is theuniversal gas constant, and [M] is the molar concentration of monovalentcations (0.07). According to this formula the nucleotide basecomposition of the oligonucleotide (contained in the terms ΔH and ΔS),the salt concentration, and the concentration of the oligonucleotide(contained in the term C) influence the T_(m). In general foroligonucleotides of the same length, the T_(m) increases as thepercentage of guanine and cytosine bases of the oligonucleotideincreases, but the T_(m) decreases as the concentration of theoligonucleotide decreases. In the case of a primer with nucleotidesother than A, T, C and G or with covalent modification, T_(m[1]), ismeasured empirically by hybridization melting analysis as known in theart.

[0011] T_(m[0]) means the T_(m) of a PCR primer or probe at the start ofa PCR amplification taking into account its starting concentration,length, and composition. Unless otherwise stated, T_(m[0]) is thecalculated T_(m) of a PCR primer at the actual starting concentration ofthat primer in the reaction mixture, under assumed standard conditionsof 0.07 M monovalent cations and the presence of a vast excessconcentration of a target oligonucleotide having a sequencecomplementary to that of the primer. In instances where a targetsequence is not fully complementary to a primer it is important toconsider not only the T_(m[0]) of the primer against its complements butalso the concentration-adjusted melting point of the imperfect hybridformed between the primer and the target. In this application, T_(m[0])for a primer is calculated using the Nearest Neighbor formula andconditions stated in the previous paragraph, but using the actualstarting micromolar concentration of the primer. In the case of a primerwith nucleotides other than A, T, C and G or with covalent modification,T_(m[0]) is measured empirically by hybridization melting analysis asknown in the art.

[0012] As used herein superscript X refers to the Excess Primer,superscript L refers to the Limiting Primer, superscript A refers to theamplicon, and superscript P refers to the probe.

[0013] T_(m) ^(A) means the melting temperature of an amplicon, either adouble-stranded amplicon or a single-stranded amplicon hybridized to itscomplement. In this application, unless otherwise stated, the meltingpoint of an amplicon, or T_(m) ^(A), refers to the T_(m) calculated bythe following % GC formula: T_(m) ^(A)=81.5+0.41 (% G+% C)−500/L+16.6log [M]/(1+0.7 [M]), where L is the length in nucleotides and [M] is themolar concentration of monovalent cations.

[0014] T_(m[0]) ^(P) refers to the concentration-adjusted meltingtemperature of the probe to its target, or the portion of probe thatactually is complementary to the target sequence (e.g., the loopsequence of a molecular beacon probe). In the case of most linearprobes, T_(m[0]) ^(P) is calculated using the Nearest Neighbor formulagiven above, as for T_(m[0]), or preferably is measured empirically. Inthe case of molecular beacons, a rough estimate of T_(m[0]) ^(P) can becalculated using commercially available computer programs that utilizethe % GC method, see Marras, S. A. et al. (1999) “Multiplex Detection ofSingle-Nucleotide Variations Using Molecular Beacons,” Genet. Anal.14:151-156, or using the Nearest Neighbor formula, or preferably ismeasured empirically. In the case of probes having non-conventionalbases and for double-stranded probes, T_(m[0]) ^(P) is determinedempirically.

[0015] C_(T) means threshold cycle and signifies the cycle of areal-time PCR amplification assay in which signal from a reporterindicative of amplicons generation first becomes detectable abovebackground. Because empirically measured background levels can beslightly variable, it is standard practice to measure the C_(T) at thepoint in the reaction when the signal reaches 10 standard deviationsabove the background level averaged over the 5-10 preceding thermalcycles.

[0016] As used herein, the terms “hybridize” or “hybridization” areart-known and include the hydrogen bonding of complementary DNA and/orRNA sequences to form a duplex molecule. As used herein, hybridizationtakes place under conditions that can be adjusted to a level ofstringency that reduces or even prevents base-pairing between a firstoligonucleotide primer or oligonucleotide probe and a target sequence,if the complementary sequences are mismatched by as little as onebase-pair. Thus, the term “stringent conditions” for hybridizationincludes conditions that minimize or prevent base-pairing between anoligonucleotide primer or oligonucleotide probe and another sequence ifthe complementary sequences are mismatched. When hybridization probessuch as differently-labeled sequence-specific molecular beacon probesare used under stringent conditions they are observed to be“allele-discriminating” because mismatches as little as one base-pairare sufficient to destabilize hybridization of the incorrect probe. Inthe context of real-time PCR “allele discrimination” is achieved bycareful attention to the design of the probe, the concentration ofmagnesium, and the temperature at which it hybridizes to its target.Single base pair mismatches between the loop sequence of the probe andits target sequence tend to have greater destabilizing effects in thecase of molecular beacons with short rather than long loop sequences.For this reason molecular beacons with short rather than long loopsequences are usually more “allele discriminating.”

[0017] As used herein “amplification target sequence” for PCRamplification means a DNA sequence that provides a template for copyingby the steps of PCR. An amplification target sequence may besingle-stranded or double-stranded. If the starting material is RNA, forexample messenger RNA, the DNA amplification target sequence is createdby reverse transcription of RNA to create complementary DNA, and theamplification target sequence is a cDNA molecule. Thus, in a PCR assayfor RNA a hybridization probe signals copying of a cDNA amplificationtarget sequence, indirectly signifying the presence of the RNA whosereverse transcription produced the cDNA molecules containing theamplification target sequence. An amplification target sequence isbracketed in length by the pair of primers used to amplify it. Therewill be a small amount of longer extension product, as explained inMullis U.S. Pat. No. 4,683,202, that is not exponentially amplified, butthe extension product of interest, whether double-stranded or, innon-symmetric PCR, single-stranded, is the exponentially amplifiedsequence, the amplicon, bracketed by the primer pair. An amplificationtarget sequence may be a single sequence. However, in some cases anamplification target sequence will contain allelic variations and, thus,not be a single sequence, even though amplified by a single primer pair.An assay for an amplification target sequence containing variations mayutilize one detector probe for all variations, a singleallele-discriminating probe for one variant, or multipleallele-discriminating probes, one for each variant.

[0018] As used interchangeably herein, the terms “nucleic acid primer”,“primer molecule”, “primer”, and “oligonucleotide primer” include short(usually between about 16 and about 50 bases) single-strandedoligonucleotides which, upon hybridization with a corresponding templatenucleic acid molecule, serve as a starting point for synthesis of thecomplementary nucleic acid strand by an appropriate polymerase molecule.Primer molecules may be complementary to either the sense or theanti-sense strand of a template nucleic acid molecule. A primer may becomposed of naturally occurring or synthetic oligonucleotides, or amixture of the two. If the primers in a pair of PCR primers are used inunequal concentrations, the primer added at the lower concentration isthe “Limiting Primer”, and the primer added at the higher concentrationis the “Excess Primer.”

[0019] As used interchangeably herein, the terms “nucleic acid probe”,“probe molecule”, and “oligonucleotide probe” and “hybridization probe”include defined nucleic acid sequences complementary to a target nucleicacid sequence to be detected such that the probe will hybridize to thetarget. Probes are typically detectably labeled, such that thehybridization of the probe to the target sequence may be readilyassessed. Probes can be composed of naturally occurring or syntheticoligonucleotides and include labeled primers. Some hybridization probes,for example molecular beacon probes, emit a detectable signal uponhybridizing to their complementary sequence without enzymatic action tohydrolyze the probes to generate a signal. We refer to such probes asprobes that hybridize to their target and “signal upon hybridization.”Other probes, for example TaqMan™ dual fluorescently labeled random coilprobes are cut, or hydrolyzed, during the amplification reaction, andhydrolysis leads to a signal change, which is detected. Probes that relyon hydrolysis as part of signal generation are not probes that “signalupon hybridization.”

[0020] A “molecular beacon probe” is a single-stranded oligonucleotide,typically 25-35 bases-long, in which the bases on the 3′ and 5′ ends arecomplementary, typically for 5-8 bases. A molecular beacon probe forms ahairpin structure at temperatures at and below those used to anneal theprimers to the template (typically below about 60° C.). Thedouble-helical stem of the hairpin brings a fluorophore attached to the5′ end of the probe very close to a quencher attached to the 3′ end ofthe probe. The probe does not fluoresce in this conformation. If a probeis heated above the temperature needed to melt the double stranded stemapart, or the probe is allowed to hybridize to a target oligonucleotidethat is complementary to the sequence within the single-strand loop ofthe probe, the fluorophore and the quencher are separated, and theresulting conformation fluoresces. Therefore, in a series of PCR cyclesthe strength of the fluorescent signal increases in proportion to theamount of the beacon hybridized to the amplicon, when the signal is readat the annealing temperature. Molecular beacons with different loopsequences can be conjugated to different fluorophores in order tomonitor increases in amplicons that differ by as little as one base(Tyagi, S. and Kramer, F. R. (1996) “Molecular Beacons: Probes ThatFluoresce Upon Hybridization,” Nat. Biotech. 14:303-308; Tyagi, S. etal., (1998) “Multicolor Molecular Beacons for Allele Discrimination.”Nat. Biotech. 16: 49-53; Kostrikis, L. G. et al., (1998) “SpectralGenotyping of Human Alleles,” Science 279: 1228-1229).

[0021] As used herein, the term “detectable label” includes moietiesthat provide a signal that may be readily detected and, in someembodiments, quantified. Such labels are well known to those in the artand include chemiluminescent, radioactive, metal ion, chemical ligand,fluorescent, or colored moieties, or enzymatic groups which, uponincubation with an appropriate substrate, provide a chemiluminescent,fluorescent, radioactive, electrical, or colorimetric signal. Methods ofdetection of such signals are also well known in the art.

[0022] As used herein, the term “buffer” includes compounds that act tomaintain the pH of a solution by maintaining the relative levels ofhydrogen and hydroxyl ions in the solution. Buffers have specific pHranges at which they are functional, and their function is frequentlytemperature-dependent. Buffers and the temperature-dependence of thebuffering capacity thereof are well known to those skilled in the art.

[0023] As used herein, the term “real time”, with respect to anamplification reaction, refers to the method by which the amplificationreaction is detected. In a “real-time” amplification reaction,accumulation of amplicon or product is measured during the progressionof the reaction, as opposed to solely after the reaction is complete,the latter being “end-point” analysis.

[0024] As used herein, the term “Optimal Annealing Temperature” is thehighest temperature at which the exponential phase of the reactionproceeds with maximal efficiency and without generating substantialnon-specific products at specific reagent concentrations and cyclingtimes. By “maximum efficiency” we mean the condition that generates thelowest C_(T) value during the exponential phase of reaction, wherein thespecific product accumulates at the highest rate.

[0025] This invention includes an amplification method that we refer toas “Linear-After-The Exponential PCR” or, for short, “LATE-PCR.”LATE-PCR is a non-symmetric PCR method; that is, it utilizes unequalconcentrations of primers and yields single-stranded primer-extensionproducts, or amplicons. LATE-PCR includes innovations in primer design,in temperature cycling profiles, and in hybridization probe design.Being a type of PCR process, LATE-PCR utilizes the basic steps of strandmelting, primer annealing, and primer extension by a DNA polymerasecaused or enabled to occur repeatedly by a series of temperature cycles.In the early cycles of a LATE-PCR amplification, when both primers arepresent, LATE-PCR amplification amplifies both strands of a targetsequence exponentially, as occurs in conventional symmetric PCR.LATE-PCR then switches to synthesis of only one strand of the targetsequence for additional cycles of amplification. In preferred real-timeLATE-PCR assays according to this invention, the Limiting Primer isexhausted within a few cycles after the reaction reaches its C_(T)value, and in the most preferred assays one cycle after the reactionreaches its C_(T) value. As defined above, the C_(T) value is thethermal cycle at which signal becomes detectable above the empiricallydetermined background level of the reaction. Whereas a symmetric PCRamplification typically reaches a plateau phase and stops generating newamplicons by the 50^(th) thermal cycle, LATE-PCR amplifications do notplateau and continue to generate single-stranded amplicons well beyondthe 50^(th) cycle, even through the 100^(th) cycle. LATE-PCRamplifications and assays typically include at least 60 cycles,preferably at least 70 cycles when small (10,000 or less) numbers oftarget molecules are present at the start of amplification.

[0026] With exceptions and limitations to be described, the ingredientsof a reaction mixture for LATE-PCR amplification are the same as theingredients of a reaction mixture for a corresponding symmetric PCRamplification. The mixture typically includes each of the fourdeoxyribonucleotide 5′ triphosphates (dNTPs) at equimolarconcentrations, a thermostable polymerase, a divalent cation, and abuffering agent. As with symmetric PCR amplifications, it may includeadditional ingredients, for example reverse transcriptase for RNAtargets. Non-natural dNTPs may be utilized. For instance, dUTP can besubstituted for dTTP and used at 3 times the concentration of the otherdNTPs due to the less efficient incorporation by Taq DNA polymerase.

[0027] As used herein, the term “Low-T_(m) Probe” means a labeledhybridization probe that signals upon hybridization to its target, whichin a LATE-PCR is the Excess Primer-Strand generated by extension of theExcess Primer, and that has a T_(m[0]) ^(P) at least 5° C. below andmore preferably at least 10° C. below the T_(m[0]) of the primer thathybridizes to and extends along the Excess Primer-Strand, which in aLATE-PCR is the Limiting Primer.

[0028] As used herein, the term “Super-Low-T_(m) Probe” means a labeledhybridization probe that signals upon hybridization to its target, whichin a LATE-PCR is the Excess Primer-Strand generated by extension of theExcess Primer (that is, a Low-T_(m) Probe), and that has a T_(m[0]) ^(P)that is at least 5° C. below, and more preferably 10° C. below the meanannealing temperature of the exponential phase of the reaction.

[0029] T_(m[1]) for a PCR primer is calculated at standard conditions ofprimers concentration and salt concentration. We have chosen 1 μM as thestandard concentration of the primers, because that concentration is atypical concentration for the Excess Primer in a LATE-PCR amplification.In LATE-PCR amplifications the Limiting Primer concentration istypically many fold times less than that standard concentration.Lowering the concentration of the Limiting Primer lowers its meltingtemperature, T_(m[0]) ^(L), in a reaction mixture. Thus, a matchedprimer pair for symmetric PCR, having equal T_(m[1])'s, will not havematched T_(m[0])'s when used at unequal concentrations. As a rule ofthumb, a primer pair that is perfectly matched, that is, for which(T_(m[1]) ^(L)−T_(m[1]) ^(X)) is zero, (T_(m[0]) ^(L)−T_(m[0]) ^(X))will be less than zero at primer concentrations used for LATE-PCR. Ourobservation is that primer pairs having equivalent initialconcentration-adjusted melting temperature at the start of the reaction,i.e. (T_(m[0]) ^(L)−T_(m[0]) ^(X))=0, will according to this inventionhave a difference in their standard calculated melting temperatures, forexample (T_(m[1]) ^(L)−T_(m[1]) ^(X)) in the range of +5-to-+20° C.(e.g., about 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 11° C.,12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C. or 20°C.).

[0030] For amplifications according to this invention the starting molarconcentration of one primer, the “Limiting Primer”, is less than thestarting molar concentration of the other primer, the “Excess Primer.”The ratio of the starting concentrations of the Excess Primer and theLimiting Primer is at least 5: 1, preferably at least 10:1, and morepreferably at least 20:1. The 5 ratio of Excess Primer to LimitingPrimer can be 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1,55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1, mostpreferably in the range of 20:1 to 100:1. Primer length and sequence areadjusted or modified, preferably at the 5′ end of the molecule, suchthat the concentration-adjusted melting temperature of the LimitingPrimer at the start of the reaction, T_(m[0]) ^(L), is greater than orequal (±0.5° C.) to the concentration-adjusted melting point of theExcess Primer at the start of the reaction, T_(m[0]) ^(X). Preferablythe difference (T_(m[0]) ^(L)−T_(m[0]) ^(X)) is at least +3, and morepreferably the difference is at least +5° C.

[0031] Amplifications according to this invention can be used togenerate single-stranded products for further use, for example asstarting material for DNA sequencing or as probes in other reactions, orcan be used in assays, including quantitative real-time assays, ofspecific nucleic acid sequences. In all cases there is a relationshipbetween T_(m[0]) ^(X) and T_(m) ^(A), which LATE-PCR takes into account.T_(m) A is higher than T_(m[0]) ^(X), but if the difference betweenthese two values is too great, lower amounts of single-stranded productwill be generated. In the case of reactions designed to generateproducts for subsequent use or analysis (T_(m) ^(A)−T_(m[0]) ^(X))should be less than or equal to 18° C., preferably not more that 15° C.For real-time assays that employ non-hydrolyzing probes, (T_(m)^(A)−T_(m[0]) ^(X)) should in all cases be less that 25° C., preferablyless than 20° C., and more preferably less than 18° C.

[0032] Amplifications and assays according to this invention can beperformed with initial reaction mixtures having ranges of concentrationsof target molecules and primers. LATE-PCR assays according to thisinvention are particularly suited for amplifications that utilize smallreaction-mixture volumes and relatively few molecules containing thetarget sequence, sometimes referred to as “low copy number.” WhileLATE-PCR can be used to assay samples containing large amounts oftarget, for example up to 10⁶ copies of target molecules, our preferredrange is a much smaller amount, from to 1-50,000 copies, more preferably1-10,000 copies and even more preferably 1-1,000 copies. Theconcentration of Limiting Primer should be from a few nanomolar (nM) upto 200 nM. The Limiting Primer concentration is preferably as far towardthe low end of the range as detection sensitivity permits. Our preferredrange with probes and detections available to us, as described below, is20-100 nM.

[0033] As with PCR, either symmetric or asymmetric, LATE-PCRamplifications according to this invention include repeated thermalcycling through the steps of strand melting, primer annealing and primerextension. Temperatures and times for the three steps are typically, aswith symmetric PCR, 93-95° C. for at least 5 sec for strand melting,55-65° C. for 10-60 sec for annealing primers, and 72° C. for 15-120 secfor primer extension. For 3-step PCR amplifications according to thisinvention, primer annealing times longer than 30 sec are not preferred.Our more preferred range is 10-20 sec. Variations of temperature andtime for PCR amplifications are known to persons skilled in the art andare generally applicable to LATE-PCR as well. For example, so-called“2-step” PCR, in which one temperature is used for both primer annealingand primer extension, can be used for LATE-PCR. In the case of “2-step”reactions the combined annealing-extension step can be longer than 30sec, but preferably as short as possible and not longer that 120 sec.

[0034] An aspect of this invention is a non-symmetric polymerase chainreaction (PCR) method comprising repeatedly thermally cycling a PCRreaction mixture containing a deoxyribonucleic acid (DNA) targetsequence, a pair of PCR primers, dNTP's and a thermostable polymerasethrough PCR steps of strand melting, primer annealing and primerextension, wherein, at the outset (a) the reaction mixture contains upto 1,000,000 copies of the nucleic acid target, (b) the PCR primer paircomprises a Limiting Primer and an Excess Primer, the Limiting Primerbeing present at a concentration of up to 200 nM and the Excess Primerbeing present at a concentration at least five times higher than theLimiting Primer, (c) the initial, concentration-adjusted meltingtemperature of the Limiting Primer is equal to or greater than theinitial, concentration-adjusted melting temperature of the ExcessPrimer, (d) the concentration-adjusted melting temperature of thatportion of the Limiting Primer which hybridizes to said target sequenceis not more than 5° C. below the concentration-adjusted meltingtemperature of the Excess Primer, (e) the melting temperature of theamplicon produced by extension of the Excess Primer exceeds the initialconcentration-adjusted melting temperature of the Excess Primer by notmore than 18° C., and (i) thermal cycling is repeated a number of timessufficient to include multiple cycles of linear amplification using theExcess Primer following exhaustion of the Limiting Primer. The foregoingmethod may also be for two or more target sequences wherein the reactionmixture includes a pair of PCR primers for each target. The method mayalso include reverse transcribing a ribonucleic acid (RNA) molecule togenerate the DNA target sequence.

[0035] Another aspect of this invention is the amplification methoddescribed above applied to low copy numbers of target, wherein thereaction mixture contains only 50,000 copies of the nucleic acid targetor even 10,000 copies, 1000 copies or one copy, or DNA or cDNA from asingle cell.

[0036] Another aspect of this invention is the amplification methoddescribed above wherein the initial, concentration-adjusted meltingtemperature of the Limiting Primer is 3-10° C. higher than the initial,concentration-adjusted melting temperature of the Excess Primer and,optionally but preferably, wherein the Excess Primer is present at aconcentration of 500-2000 nM and at least ten times higher than theLimiting Primer, and, also optionally but preferably, wherein themelting temperature of the amplicon is 7-15° C. higher than the initial,concentration-adjusted melting temperature of the Excess Primer.

[0037] Another aspect of this invention is the method described abovewherein the duration of the primer annealing step is not longer than 30seconds.

[0038] Another aspect of this invention is a variant of the methoddescribed above further including at least one terminal thermal cycle inwhich the single-stranded extension product of the Excess Primer isconverted to double-stranded product, by including in the PCR reactionmixture a low-temperature primer capable of priming the extensionproduct of the Excess Primer and having an initial,concentration-adjusted melting point at least 5° C. below, morepreferably at least 10° C. below, the initial, concentration-adjustedmelting point of the Excess Primer, and wherein the annealingtemperature is maintained above the initial, concentration-adjustedmelting temperature of the low-temperature primer except for at leastone terminal cycle in which the annealing temperature is lowered tohybridize the low-temperature primer.

[0039] Another aspect of this invention is a non-symmetric polymerasechain reaction (PCR) method comprising thermally cycling a PCR reactionmixture containing a deoxyribonucleic acid (DNA) target sequence, a pairof matched Limiting PCR Primers, an additional Excess Primer, dNTP's anda thermostable polymerase repeatedly through PCR steps of strandmelting, primer annealing and primer extension, wherein the matched PCRprimers are present in approximately equimolar concentration of up to200 nM, the Excess Primer is present at a concentration at least fivetimes higher than the Limiting Primers, the initial,concentration-adjusted melting temperatures of the Excess Primers is atleast 5° C. below, more preferably at least 10° C. below, theconcentration-adjusted melting temperatures of the Limiting Primers, andwherein the reaction comprises a first phase wherein the annealingtemperature is higher than the initial, concentration-adjusted meltingtemperature of the Excess Primer, and the matched Limiting Primersgenerate a first amplicon, and a second phase wherein the annealingtemperature is lowered and the Excess Primer generates a secondamplicon, shorter than the first amplicon, utilizing the first ampliconas a template strand, and wherein the melting temperature of the secondamplicon exceeds the initial, concentration-adjusted melting temperatureof the Excess Primer by not more than 25° C., more preferably by notmore than 18° C.

[0040] Another aspect of this invention is a non-symmetric polymerasechain reaction (PCR) method with removal of single-stranded ampliconcomprising

[0041] a) thermally cycling a PCR reaction mixture containing a DNAtarget, a pair of PCR primers for said target, dNTP's and a thermostableDNA polymerase through repeated cycles of strand melting, primerannealing and primer extension, wherein (i) the PCR primer paircomprises a Limiting Primer and an Excess Primer, (ii) the LimitingPrimer is present at a concentration of up to 200 nM, and the ExcessPrimer is present at a concentration at least five times higher than theLimiting Primer, (iii) the initial, concentration-adjusted meltingtemperature of the Limiting Primer is at least equal to, more preferably3-10° C. higher than, the initial, concentration-adjusted meltingtemperature of the Excess Primer, and (iv) thermal cycling is repeated anumber of times sufficient to include multiple cycles of linearamplification using the Excess Primer following exhaustion of theLimiting Primer; and

[0042] b) during at least the cycles of linear amplification, followingthe step of primer extension permanently removing the single-strandedextension product of the Excess Primer from the reaction mixture byhybridizing said product to immobilized capture probes. In preferredversions of the method the immobilized capture probes are in a thermallyisolated product removal zone and said step of removing comprisespassing the reaction mixture through said zone. In certain preferredversions of this method the capture probes are isolatable (for example,beads that can be physically removed from the reaction mixture) or arein a product removal zone that itself is physically isolatable from saidat least one reaction zone, further including periodically isolatingsaid capture probes and harvesting product hybridized to said captureprobes not in contact with the reaction mixture, such as while thereaction mixture is in said at least one reaction zone. In otherpreferred versions the melting temperature of the amplicon exceeds theconcentration-adjusted melting temperature of the Excess Primer by notmore than 18° C. In yet other preferred versions the Excess Primer ispresent at a concentration of 500-2000 nM and at least ten times higherthan the Limiting Primer.

[0043] Another aspect of this invention is a homogeneous real-timedetection assay for a DNA target sequence employing non-symmetricpolymerase chain reaction (PCR) amplification, comprising thermallycycling a PCR reaction mixture containing said target sequence, a pairof PCR primers for amplifying said target sequence, dNTP's, at least onelabeled hybridization probe that binds to the amplicon product by saidamplification, and a thermostable DNA polymerase through repeated PCRsteps of strand melting, primer annealing, and primer extension, wherein(i) the PCR primer pair comprises a Limiting Primer and an ExcessPrimer, (ii) the Limiting Primer is present at a concentration of up to200 nM, and the Excess Primer is present at a concentration of at leastfive times higher than the Limiting Primer, (iii) the initial,concentration-adjusted melting temperature of the Limiting Primer is atleast equal to the initial, concentration-adjusted melting temperatureof the Excess Primer, (iv) said probe hybridizes to said amplicon duringthe primer annealing step of PCR, (v) the melting temperature of theamplicon exceeds the initial, concentration-adjusted melting temperatureof the Excess Primer by not more than 25° C., and (vi) thermal cyclingis repeated a number of times sufficient to include multiple cycles oflinear amplification using the Excess Primer following exhaustion of theLimiting Primer, and (vii) said probe emits a detectable signalindicative of product generation during said linear amplification. Incertain versions of this assay the hybridization probe is a dual-labeledfluorescent probe that binds to the extension product of the LimitingPrimer and that is hydrolyzed by the polymerase during extension of theExcess Primer, thereby generating a detectable signal. In more preferredversions the hybridization probe (or probes) is a dual-labeledfluorescent probe that binds to the extension product of the ExcessPrimer and that signals upon hybridization, such as molecular beaconprobes, FRET probe pairs, hybridized probe pairs, and Excess Primerscontaining attached hairpin probes. Certain preferred versions include afirst probe for one allelic variant and a second probe for anotherallelic variant. The assay may include reverse transcribing ofribonucleic acid (RNA) molecules to generate cDNA containing targetsequences.

[0044] The foregoing assay may be for small copy numbers of targets,such as wherein the reaction mixture contains up to 50,000 copies of thenucleic acid target, or even 1000 copies, or one copy or cDNA from asingle cell. In certain preferred embodiments the initial,concentration-adjusted melting temperature of the Limiting Primer is3-10° C. higher than the in initial, concentration-adjusted meltingtemperature of the Excess Primer. In certain preferred embodiments theExcess Primer is present at a concentration of 500-2000 nM and at leastten times higher than the Limiting Primer. In certain preferredembodiments the melting temperature of the amplicon is 7-15° C. higherthan the initial, concentration-adjusted melting temperature of theExcess Primer. In our most preferred embodiments the duration of theprimer annealing step is not longer than 30 seconds.

[0045] LATE-PCR can be combined with the use of very bright probes, suchas probes labeled with Quantum Dots®, which permit detection of smallnumbers or very small numbers of DNA molecules, in the range of 1000 toone million single strands. As the signal strength of the probe isincreased, the number of single-stranded molecules that have to begenerated by the LATE-PCR decreases. This can be accomplished byreducing the absolute concentration of the Limiting Primer or bydecreasing the volume of the reaction at a constant limiting Primerconcentration. As the absolute concentration of the Limiting Primer isdecreased and the number of single-stranded molecules produced isdecreased, detection of smaller numbers of molecules takes place underconditions in which the Excess Primer does not have to compete withreannealing of the product single-strand to the target strand. LATE-PCRreactions carried out in the presence of bright probes is well suited tominiaturization, for instance for production of chips and chambers thatcarry out reactions using microfluidics. Therefore, the requirement that(T_(m) ^(A)−T_(m[0]) ^(X))≦25 becomes relaxed.

[0046] Another aspect of this invention is a homogeneous detection assayfor a DNA target sequence employing non-symmetric polymerase chainreaction (PCR) amplification, comprising thermally cycling a PCRreaction mixture containing said target sequence, a pair of PCR primersfor said target sequence, dNTP's, a labeled low-temperaturehybridization probe, and a thermostable DNA polymerase repeatedlythrough PCR steps of strand melting, primer annealing and primerextension, wherein (i) the PCR primer pair comprises a Limiting Primerand an Excess Primer, (ii) the Limiting Primer is present at aconcentration of up to 200 nM, and the Excess Primer is present at aconcentration of at least five times the concentration of the LimitingPrimer, (iii) the initial, concentration-adjusted melting temperature ofthe limiting primer is equal to or greater than the initial,concentration-adjusted melting temperature of the Excess Primer, (iv)the melting temperature of the amplicon exceeds the initial,concentration-adjusted melting temperature of the Excess Primer by notmore than 25° C., (v) the low-temperature hybridization probe binds tothe extension product of the Excess Primer and emits a detectable signalupon hybridization, (vi) the initial, concentration-adjusted meltingtemperature of the low-temperature hybridization probe is at least 5°below the initial, concentration-adjusted melting temperature of theLimiting Primer, (vii) thermal cycling is repeated a number of timessufficient to include multiple cycles of linear amplification using theExcess Primer following exhaustion of the Limiting Primer, and (viii)detection is performed at a temperature below said optimal annealingtemperature.

[0047] In certain embodiments of this assay the initial,concentration-adjusted melting temperature of the low-temperaturehybridization probe is at least 10° C. below the initial,concentration-adjusted melting temperature of the Limiting Primer. Insome embodiments of this assay primer annealing is of sufficiently lowtemperature and of sufficient duration that the low-temperature probehybridizes during primer annealing, the signal detection is performedduring that step. In more preferred embodiments the PCR amplificationincludes, for at least the last few cycles of exponential amplificationand the subsequent cycles of linear amplification, an added detectionstep following primer extension, said detection step being ofsufficiently low temperature and sufficient duration for thelow-temperature hybridization probe to hybridize and signal, and whereinthe PCR step of primer annealing is not of sufficiently low temperatureand/or of sufficient duration for said probe to hybridize and signal. Incertain preferred embodiments the initial concentration-adjusted meltingtemperature of the low-temperature hybridization probe is at least 5° C.below, more preferably at least 10° C. below, the temperature of theannealing step of the amplification reaction, and wherein at least thelinear amplification cycles include a low-temperature detection step,preferably of 10-30 seconds duration, following primer extension inwhich the temperature is lowered below the annealing temperature tohybridize said probe, and detection is performed. In a version of theseembodiments the PCR reaction mixture additionally includes alow-temperature masking oligonucleotide that is complementary to theExcess Primer and that has an initial, concentration-adjusted meltingpoint at least 5° C. below the initial, concentration-adjusted meltingpoint of the Excess Primer. In other versions the low-temperaturehybridization probe is a molecular beacon probe.

[0048] Another aspect of this invention is oligonucleotide setscontaining the primers or the primers and the probes for performing theforegoing amplifications and assays. Primers are preferably usedtogether in a single buffer so that the ratio of Limiting Primer toExcess Primer is fixed. Also preferably an oligonucleotide set specifiesan intended concentration of at least one primer or a mixture of thetwo, to ensure that (T_(m[0]) ^(L)−T_(m[0]) ^(X)) meets the criterion ofthe invention.

[0049] Another aspect of this invention is reagent kits for performingthe foregoing assays. The kits include, in addition to the primers andprobes, at least a DNA polymerase and dNTP's. Preferably all reagentsnecessary to perform the assay are included. However, individualreagents, such as, for example the polymerase, may be separatelypackaged. Kits should include instructions for performing particularassays.

[0050] Another aspect of this invention is a method for amplification ofa nucleic acid target sequence present in a sample containing from oneto about 10,000 copies of said target sequence, the method comprising:

[0051] a) contacting the nucleic acid target sequence with a firstoligonucleotide primer and a second oligonucleotide primer, wherein theT_(m) of the first primer is at least 5° C. greater, preferably 10° C.or even 20° C. greater, than the T_(m) of the second primer and whereinthe concentration of the second primer is up to 1000 nM and at leastabout 10 times greater, or even 20-100 times greater, than theconcentration of the first primer; and

[0052] b) amplifying the target sequence by a polymerase chain reactionutilizing said first and second oligonucleotide primers, said reactionhaving an exponential phase of amplicon generation followed by a linearphase of amplicon generation that utilizes only the second primer.

[0053] Another aspect of this invention is a method for detecting atleast one nucleic acid sequence in a sample containing up to about10,000 copies of said at least one nucleic acid sequence, the methodcomprising:

[0054] a) contacting the at least one nucleic acid target sequence witha first oligonucleotide primer hybridizable thereto and a secondoligonucleotide primer hybridizable thereto, wherein the T_(m) of thefirst primer is at least 5° C. greater, preferably 10° C. to 20° C.greater, than the T_(m) of the second primer and wherein theconcentration of the second primer is up to 1000 nM and at least about10 times greater than the concentration of the second primer;

[0055] b) amplifying the at least one target sequence by a polymerasechain reaction utilizing said first and second oligonucleotide primers,said reaction having an exponential phase of amplicon generationfollowed by a linear phase of amplicon generation that utilizes only thesecond primer; and

[0056] c) detecting amplicon generated from said second primer in realtime during the polymerase chain reaction by means of a firsthybridization probe targeted thereto. The nucleic acid sequence may be agenetic sequence subject to allelic mutation wherein said firsthybridization probe is targeted to a first allelic variant. Amplicongenerated from said second primer may be detected in real time duringthe polymerase chain reaction by means of a second hybridization probetargeted to a second allelic variant. In certain embodiments theconcentration of the second primer is 20-100 times the concentration ofthe first primer. This method may be utilized to detect at least twodifferent nucleic acid sequences, in which case the method comprisescontacting each nucleic acid sequence with a first primer hybridizablethereto and a second primer hybridizable thereto. In certain preferredembodiments detection is performed between the PCR steps of primerextension step and strand melting step, preferably at a temperaturebelow the primer extension temperature. The probe may be a molecularbeacon probe or a double strand probe, among others.

[0057] Another aspect of this invention is a composition comprising atleast one pair of polymerase chain reaction primers for at least onepre-selected nucleic acid target sequence, said at least one paircomprising a first primer and a second primer, wherein the T_(m) of thefirst primer is at least 5° C. greater, preferably 10-20° C. greater,than the T_(m) of the second primer and wherein the concentration of thesecond primer is at least 10 times greater, preferably 20-100 timesgreater, than the concentration of the first primer. Embodiments of thisaspect of the invention include at least one hybridization probe,preferably one is targeted against the extension product of the secondprimer. The probe may be a molecular beacon probe.

[0058] Another aspect of this invention is a kit of reagents forperforming a real-time polymerase chain reaction assay for at least onepre-selected nucleic acid target sequence, comprising at least one pairof polymerase chain reaction primers including a first primer and asecond primer, four deoxyribonucleotide triphosphates, a thermostableDNA polymerase, and a labeled hybridization probe that emits adetectable signal upon hybridization, wherein

[0059] a) the T_(m) of the first primer is at least 5° C. greater,preferably 10-20° C. greater, than the T_(m) of the second primer andthe concentration of the second primer is at least 10 times greater,preferably 20-100 times greater, than the concentration of the secondprimer, and

[0060] b) said labeled hybridization probe, which may be a molecularbeacon, is targeted against the extension product of said second primer.

[0061] This invention also includes assays utilizing LATE-PCRamplification, including both end-point assays, which may or may not behomogeneous assays, and homogeneous real-time assays utilizing labeledhybridization probes (including labeled primers) that produce a signalchange due to extension of the Excess Primer to make single-strandedamplicons during the later cycles of amplification. Detection methodsknown for PCR can be applied to LATE-PCR assays.

[0062] Preferred LATE-PCR assays utilize labeled hybridization probesthat are complementary to a sequence in the single-stranded ampliconproduced by extension of the Excess Primer and emit a detectable signalupon hybridization. During the latter phase of LATE-PCR, whensingle-stranded amplicon is being produced, that single strand does notserve as a template strand. Hence, while TaqMan™ dual-labeled probesthat are cut by DNA polymerase during primer extension can be used inLATE-PCR assays, they are not suitable to measure single-strandedproduct directly. Probes such as molecular beacon probes,double-stranded probes, and FRET hybridization probes are suitable forthat purpose. In homogeneous, real-time LATE-PCR assays, probes thattarget the single-stranded amplicon are present in the initial reactionmixture. During the exponential phase of the amplification such probestarget the same amplicon strand as does the Limiting Primer.

[0063] A further embodiment of the invention is to use Low-T_(m)hybridization probes. The T_(m[0]) ^(P) of Low-T_(m) probes is equal toor below, preferably at least 5° C. below, most preferably at least 10°C. below, the T_(m[0]) ^(L) of the Limiting Primer. Low-T_(m) Probesused in a LATE-PCR can either be detected during the annealing step of a2 or 3 step reaction, or can be detected during a step added after theextension step and prior to the next melting step. These probes have theadded benefit of being more allele discriminating than conventionalhybridization probes.

[0064] A preferred embodiment of the invention uses Super-Low-T_(m)hybridization probes. The T_(m[0]) ^(P) of a Super-Low-T_(m) probe is atleast 5° C. below, and more preferably 10° C. below the mean annealingtemperature of the reaction. Super-Low-T_(m) probes are preferablyemployed in LATE-PCR assays in conjunction with the novel detectionstep, described above, which is carried out under preferred conditionsof lowered temperature to accommodate the properties of such probes. Ifa constant temperature is used for annealing step throughout theexponential phase of the reaction, T_(m[0]) ^(P) is at least 5° C., mostpreferably at least 10° C., below that temperature. If the annealingtemperature is not constant during the cycles of the exponential phaseof the reaction the preferred temperature in this case is at least 5°C., most preferred at least 10° C. below the mean annealing steptemperature of the exponential phase of the reaction. Like Low-T_(m)probes, Super-Low-T_(m) probes are more allele discriminating thanconventional hybridization probes.

[0065] Certain preferred embodiments utilize an added detection step inall or, preferably only some, amplification cycles. The detection stepis of minimal duration, generally 10-30 seconds, sufficient for probesto hybridize and signal. We prefer to utilize this step beginning 5-10cycles prior to the anticipated threshold cycle, C_(T). We also preferto utilize this added detection step following the primer-extensionstep. This step effectively separates primer-annealing-and-extensionfrom probe-annealing-and-detection.

[0066] Certain preferred embodiments utilize a low temperature versionof the added detection step in all or some amplification cycles, namely,a low-temperature detection step, which comprises dropping thetemperature below the temperature of the previous annealing step for atime sufficient for Low-T_(m) probes to hybridize and signal, generally10-30 sec. We prefer to utilize this step beginning 5-10 cycles prior tothe anticipated threshold cycle, C_(T). We also prefer to utilize thisadded detection step to follow the extension step. In other embodimentslow-temperature detection is performed after strand melting but beforeprimer extension. After low-temperature detection the temperature israised either to the strand-melting temperature or to theprimer-extension temperature.

[0067] In preferred embodiments the probes are not hybridized to targetand amplicon strands during primer annealing and primer extension, anddetection is uncoupled from primer annealing. In contrast, conventionalreal-time PCR assays of the prior art that utilize probes that signalupon hybridization, such as molecular beacon probes, hybridize bothprimers and probes during the annealing step, and assays rely on atemperature rise for primer extension to remove probes but not primersfrom template strands.

[0068] This invention also includes sets of primers and probes forperforming LATE-PCR amplifications and assays. We sometimes refer tothese as “oligonucleotide sets.” The set for an amplification or assayincludes one or more pairs of an Excess Primer and a Limiting Primerhaving melting temperatures and concentration ratios as describedherein. Embodiments for real-time assays further include at least onelabeled hybridization probe as described herein, including for certainpreferred embodiments Low-T_(m) hybridization probes or Super-Low-T_(m)hybridization probes that only hybridize during the low-temperaturedetection step described above. An oligonucleotide set containing oneprimer pair may contain more than one probe, for instance, one for thewild-type sequence being amplified and one for its mutant allele. Foroligonucleotide sets one can include the primers or the primers andprobes in separate buffers or, as is preferred, in a single buffer tofix their concentration ratios. Oligonucleotide sets are designedaccording to the principles of LATE-PCR and thus have specified, or“intended” concentrations, rations and initial, concentration-adjustedmelting temperatures. For example, the intended, concentration-adjustedmelting temperature of the Limiting Primer should be at least equal tothe intended concentration-adjusted melting temperature of the ExcessPrimer, and so on.

[0069] This invention also includes reagent kits for amplifications andassays. Kits for amplification include, in addition to primer sets, atleast a DNA polymerase, four dNTP's and amplification buffer. Real-timehomogeneous assay kits contain oligonucleotide sets that include primersand labeled hybridization probes as well as DNA polymerase, four dNTP'sand amplification buffer. Kits may contain additional ingredients forsample preparation, as well as controls. Complete kits contain allreagents necessary for performing a LATE-PCR amplification or assay and,optionally, disposable materials to be used. Kits may be in one packageor in multiple packages, that is, modular.

[0070] Multiplexing involves the simultaneous amplification in a singlereaction vessel of two or more target sequences utilizing multipleprimer pairs, one for each target. An aspect of this invention ismultiplex LATE-PCR assays, kits and oligonucleotide sets. In multiplexassays it is preferred that the T_(m[0]) ^(L) of all Limiting Primers inthe reaction be made equal to or greater than the T_(m[0]) ^(X) of allExcess Primers in the reaction. It is recommended that primer candidatesbe subjected to computer analysis to screen out obvious cases ofprimer-dimer formation, as well as inappropriate product strandinteractions.

[0071] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention Will be apparent fromthe description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

[0072]FIG. 1. presents real-time fluorescence curves from PCRamplifications with a primer pair according to this invention and with asymmetric PCR primer pair.

[0073]FIG. 2A presents real-time fluorescence curves from PCRamplifications with primer pairs having varying values of the difference(T_(m[0]) ^(L)−T_(m[0]) ^(X)), using a constant annealing temperature.

[0074]FIG. 2B presents real-time fluorescence curves from PCRamplifications with primer pairs having varying values of the difference(T_(m[0]) ^(L)−T_(m[0]) ^(X)), and an annealing temperature relative toT_(m[0]) ^(L).

[0075]FIGS. 3A, 3B, 3C present real time fluorescence curves fromreplicate PCR amplifications with primer pairs having various T_(m)relationships at both equal and unequal concentrations.

[0076]FIGS. 4A, 4B present real-time fluorescence curves from replicatePCR amplifications with primer pairs having different T_(m)relationships at both equal and unequal concentrations.

[0077]FIGS. 5A, 5B, 5C present real-time fluorescence curves from PCRamplifications having primer pairs having different T_(m) relationshipsat several concentration ratios.

[0078]FIG. 6 presents real-time fluorescence curves from PCRamplifications having varying values of (T_(m) ^(A)−T_(m[0]) ^(X)) with(T_(m[0]) ^(L)−T_(m[0]) ^(X)) in the range of 0.0° C.

[0079]FIG. 7 presents real-time fluorescence curves from PCRamplifications having varying values of (T_(m) ^(A)−T_(m[0]) ^(X)) with(T_(m[0]) ^(L)−T_(m[0]) ^(X)) in the range of 5-6° C.

[0080]FIG. 8 presents real-time fluorescence curves from PCRamplifications having different concentrations of Low-T_(m) Probe.

[0081]FIG. 9 presents real-time fluorescence curves from PCRamplifications of homozygous cells compared to heterozygous cells.

[0082]FIG. 10 presents real-time fluorescence curves from a multiplexPCR amplification of two target sequences.

[0083]FIGS. 11A, 11B present melt analyses and fluorescence curves fromLATE-PCR amplifications performed with a stringent annealing step andwith a non-stringent annealing step.

[0084] Like reference symbols in the various drawings indicate likeelements.

DETAILED DESCRIPTION

[0085] Designing Limiting and Excess Primer Pairs

[0086] Design of primer pairs for use in this invention can be performeddirectly, as will be explained. Alternatively, it can begin withselecting or designing a primer pair for symmetric PCR by known methods,followed by modifications for LATE-PCR. Symmetric PCR primers aredesigned to have equal melting points at some set of standard conditionsof primers concentration and salt concentration. Symmetric PCR primersare conveniently designed and analyzed utilizing an available computerprogram. For symmetric and asymmetric PCR the standard techniques forcalculating melting temperatures (T_(m)) have been the “NearestNeighbor” method and the “2(A+T)+4(G+C)” method. For clarity weintroduce the concept of T_(m[1]) which is the T_(m) of the primer at astandard primer concentration of 1 μM and 0.07M salt (monovalentcations). Conversion from the T_(m) given by a typical computer programto T_(m[1]) generally has minimal effect on the relationship of theT_(m)'s of a primer pair. For the concentration-adjusted meltingtemperatures of primer pairs according to this invention, either actualmeasurement or an appropriate calculation is required. For the purposeof describing and comparing primer melting points according to thisspecification and claims, “concentration-adjusted melting point,” or“T_(m[0])”, we calculate the melting point according to the NearestNeighbor formula set forth above in the definitions used in thisspecification where possible; otherwise we determine T_(m[0])empirically.

[0087] In practice, once a particular target sequence (for instance asequence flanking a mutation within a gene) has been chosen foramplification, several candidate pairs of equal T_(m) primers aredesigned via a computer program such as Oligo 6.0® using the program'sdefault values. The candidate primer pairs are then scrutinized on thebasis of additional criteria, such as possible primer-dimer formation,that are known in the art to cause non-desirable primer qualities.Satisfactory pairs of candidate primers are further scrutinized usingsoftware such as “Blast” for possible non-specific matches to DNAsequences elsewhere in the known genome from the species of the targetsequence (Madden, T. L. et al. (1996) “Applications of Network BLASTServer,” Meth. Enzymol. 266: 131-141). Primers pairs are then comparedas to their T_(m[0]) values at several different possible concentrationsand ratios such that the primer chosen to be the Limiting Primer willhave an equal or greater T_(m[0]) relative to the primer chosen to bethe Excess Primer. In addition, pairs of candidate primers are examinedin relation to the sequence of the amplicon they are expected togenerate. For instance, certain target sequences may contain a GC-richsequence at one end and a less GC-rich sequence at the other end. Wherethat occurs, choosing the Limiting Primer sequence within sequences atthe GC-rich end will assist in achieving a higher melting point for theLimiting Primer relative to the Excess Primer, which will consists ofsequences in the less GC-rich end. Examination of the candidate primerpairs relative to the amplicon sequence may suggest additional or novelways of modifying the sequences of one or both members of the pair, suchas deliberately increasing or decreasing the length of the primer, mostpreferably at its 5′ end, or introducing changes in base sequenceswithin the primer which deliberately cause it to mismatch with itstarget in small regions. All such changes will increase or decrease theT_(m[0]) of either the Limiting or Excess primer.

[0088] Table I illustrates two possible primer pairs for one allele inthe HEX-A gene which is responsible for Tay-Sachs disease, as well asthe LATE-PCR criterion used to judge whether or not they were likely tobe suitable for LATE-PCR. In accord with the theoretic principles ofLATE-PCR, the experimental assay discussed below in connection with FIG.1 established that only the primer pair for which (T_(m[0])^(L)−T_(m[0]) ^(X))≧0 (i.e., primer set 2, Table I) was suitable forLATE-PCR. In comparing the two primer sets, it will be noted that theLimiting Primer in Set 2 has two additional 5′ nucleotides as comparedto its corresponding primer in Set 1. The Excess Primer is the same inboth sets. TABLE I Possible primers pairs and the LATE-PCR criterion fortheir selection SEQ ID (T_(m[0]) ^(L)− Sequence NO: Conc. T_(m[1]) Conc.T_(m[0]) T_(m[0]) ^(X)) Primer Set 1 TSD1242S205′-CCTTCTCTCTGCCCCCTGGT-3′ 1 1 μM 64.8° C. 25 nM 58.9° C. −5 TSD1301A225′-GCCAGGGGTTCCACTACGTAGA-3′ 2 1 μM 64.3° C.  1 μM 64.3° C. Primer Set 2TSD1240S22 5′-GCCCTTCTCTCTGCCCCCTGGT-3′ 3 1 μM 69.4° C. 25 nM 64.0° C. 0TSD1301A22 5′-GCCAGGGGTTCCACTACGTAGA-3′ 2 1 μM 64.3° C.  1 μM 64.3° C.

[0089] As explained above, in the case of LATE-PCR T_(m[1]) ^(L) must begreater than T_(m[1]) ^(X) in order to guarantee that actual meltingtemperature of the Limiting Primer is greater than or equal to theactual melting temperature of the Excess Primer at the actual primerconcentrations in the reaction, i.e. (T_(m[0]) ^(L)−T_(m[0]) ^(X))≧0. Ifthis condition is not met, i.e. (T_(m[0]) ^(L)−T_(m[0]) ^(X))<0,amplification reactions run inefficiently. These features of LATE-PCRare illustrated in FIG. 1, which shows real-time LATE-PCR reactions withthe primer pairs of Table I in which the double-stranded productssynthesized during the exponential phase of the reaction have beenvisualized using SYBR® Green. Curve 12 shows the efficient reaction(product detected in fewer thermal cycles), (T_(m[0]) ^(L)−T_(m[0])^(X))=0 and (T_(m[1]) ^(L−T) _(m[1]) ^(X))=5, while curve 11 shows theinefficient reaction (product detected in more thermal cycles),(T_(m[0]) ^(L)−T_(m[0]) ^(X))=−5 and (T_(m[1]) ^(L)−T_(m[1])^(L)−T_(m[1]) ^(X))=1. Both reactions amplified the same region of theHEX-A gene and both were initiated with 1000 genomes. In the case ofcurve 12, T_(m[1]) ^(L)=69° C. and T_(m[1]) ^(X)=64° C., while in thecase of Curve 11, T_(m[1]) ^(L)=64 while T_(m[1]) ^(X)=64° C. (see TableI). The design and execution of this experiment are described in detailin Example 1.

[0090]FIG. 2A, based on the pairs of primers described in Example 3,illustrates the fact that as T_(m[1]) ^(L) is increased several degreesabove T_(m[1]) ^(X), (T_(m[0]) ^(L)−T_(m[0]) ^(X)) becomes >0, and theefficiency of the LATE-PCR increases still further. Each of the curvesin FIG. 2A shows the mean real time fluorescence increase in sampleswith (T_(m[0]) ^(L)−T_(m[0]) ^(X))=+7 (curve 21), +5 (curve 22), +3(curve 23), 0 (curve 24), and −3 (curve 25). Each curve depicts theaverage fluorescence of 3 replicate samples. The earliest detection(lowest mean C_(T) value) was obtained using the primer pair with thehighest value (T_(m[0]) ^(L)−T_(m[0]) ^(X)), curve 21. Mean C_(T) valuesincreased with each decrease in the value of (T_(m[0]) ^(L)−T_(m[0])^(X)). Lower C_(T) values demonstrate a higher rate of amplification(i.e., increased efficiency) during the exponential phase of thereaction. Experimental details about this experiment are provided belowin Example 3. Example 3 (and FIG. 2B therein) also describe anadditional experiment illustrating that the efficiency and specificityof the LATE-PCR improves when (T_(m[0]) ^(L)−T_(m[0]) ^(X)) becomes >0.

[0091] FIGS. 3A-3C show examples of amplification using three differentCFTR primer sets. The experimental details of FIG. 3 are described inExample 4. The primers were either equimolar (both at 500 nM; curves 32,34, and 36) or present at a 1:10 ratio (50 nM Limiting Primer:500 nMExcess Primer; curves 31, 33, and 35). All experiments used a molecularbeacon probe that monitored synthesis of the Excess Primer strand of theamplicon for the cystic fibrosis ΔF508 allele. T_(m) values for theprimers were initially obtained using the default parameters of theOligo® 6.0 program. Based on this program the equimolar T_(m) values ofthe primers used in FIG. 3 were as follows: FIG. 3A both primers 65° C.;FIG. 3B Limiting Primer 70° C. and Excess Primer 65° C.; FIG. 3CLimiting primer 75° C. and Excess primer 65° C.

[0092] As shown in FIG. 3A, the asymmetric reaction (1:10 primer ratio,curve 31) using the two primers with the same T_(m)'s results in afluorescence signal that is delayed (higher C_(T)), as compared to thesymmetric reaction (equimolar primers, curve 32). However, when a 5° C.difference in T_(m)'s is introduced (FIG. 3B), the C_(T) for the primerswith a 1:10 ratio (curve 33) occurs much earlier, almost as early as forthe equimolar primers (curve 34). Additionally, the final fluorescencesignal for the primers with a 1:10 ratio (curve 33) is much higher thanthe signal for the equimolar primers (curve 34), and it has notplateaued, even beyond 60 cycles. When a 10° C. difference in T_(m)'s(FIG. 3C) is introduced, the C_(T) for the primers with a 1:10 ratio(curve 35) is the same as for the equimolar primers (curve 36), and thefinal fluorescence is much higher and does not plateau.

[0093] FIGS. 4A-4B show a similar example using two sets of primers forthe Tay-Sachs Disease, HEX-A gene. In this case the primers were eitherequimolar (both 300 nM; curves 41 and 44) or present at a 1:100 ratio(10 nM Limiting Primer; 1000 nM Excess Primer; curves 42 and 43). Allexperiments used a molecular beacon probe that monitored synthesis ofthe single-stranded product of the Excess Primer for the normal alleleof the HEX-A gene, in the region of the gene that includes 1278-diseasecausing allele. Once again, the T_(m) values from the primers wereinitially obtained using the default parameters of the Oligo® 6.0program. Based on this program the equimolar T_(m) values of the primersused in FIG. 4 were as follows: FIG. 4A both primers 72° C.; FIG. 4BLimiting Primer 84° C. and Excess Primer 72° C.

[0094] Once again, the asymmetric reaction (1:100 primer ratio, curve42) using equal T_(m) primers (as calculated by the default values ofthe Oligo® 6.0 program) results in a fluorescence signal that is delayed(higher C_(T)), as compared to the symmetric reaction (equimolarprimers, curve 42, FIG. 4A). However, when a 12° C. difference inT_(m)'s is introduced (FIG. 4B), the C_(T) for the primers with a 1:100ratio (curve 43) is the same as for the equimolar primers (curve 44),and the final fluorescence is much higher and does not plateau.

[0095] Application of the “Nearest Neighbor” formula allows the defaultT_(m) values obtained from the Oligo® 6.0 software to be converted intoT_(m[0]) values that take the actual starting concentration of eachprimer into account, as shown in Table II. The T_(m) values calculatedby Oligo 6.0 are useful only as a rough approximation, since they arebased on the thermodynamic values and salt correction factors ofBreslauer et al. (Breslauer K J et al., (1986) “Predicting DNA DuplexStability From The Base Sequence, “Proc. Natl. Acad. Sci. USA 83:3746-50), and are relatively inaccurate (Owczarzy R, et al., (1998)“Predicting Sequence-Dependent Melting Stability of Short Duplex DNAOligomers,” Biopolymers 44: 217-39; SantaLucia J. (1998) “A Unified Viewof Polymer, Dumbbell, and Oligonucleotide DNA Nearest-NeighborThermodynamics,” Proc. Natl. Acad. Sci. USA 95: 1460-5). The resultingdata fully account for the results shown in FIGS. 3 and 4 in terms ofthe principles of LATE-PCR. Only the reactions illustrated in FIG. 3Cand 4B meet the requirement that (T_(m[0]) ^(L)−T_(m[0]) ^(X))≧0, andtherefore are LATE-PCR reactions. Only these reactions have the lowestC_(T) values and highest final fluorescence signals and do not plateaulike reactions utilizing equimolar primers. In contrast, theconventional asymmetric reactions in FIGS. 3A, 3B, and 4A have (T_(m[0])^(L)−T_(m[0]) ^(X))<0. These reactions are inefficient (higher C_(T)values and lower fluorescence). TABLE II T_(m[0]) values for the CFTRand HEX-A primers in FIGS. 3 and 4 Primer Primer ratio SEQ ratio (equal)(unequal) Criteria ID Conc. T_(m[0]) Conc. T_(m[0]) (T_(m[0]) ^(L) −Primers Sequence NO: (nM) (° C.) (nM) (° C.) T_(m[0]) ^(X)) FIG. 3ACF403S18 5′GATTATGCCTGGCACCAT3′ 4 500 55.0 50 52.5 −3.0 CF482A235′CTTTGATGACGCTTCTGTATCTA3′ 5 500 55.5 500 55.5 FIG. 3B CF402S195′GGATTATGCCTGGCACCAT3′ 6 500 57.9 50 54.1 −1.4 CF482A235′CTTTGATGACGCTTCTGTATCTA3′ 5 500 55.5 500 55.5 FIG. 3C CF399S225′CCTGGATTATGCCTGGCACCAT3′ 7 500 62.8 50 59.5 +4.0 CF482A235′CTTTGATGACGCTTCTGTATCTA3′ 5 500 55.5 500 55.5 FIG. 4A TSD1242S205′-CCTTCTCTCTGCCCCCTGGT-3′ 1 300 62.8 10 58.9 −5.4 TSD1301A225′-GCCAGGGGTTCCACTACGTAGA-3′ 2 300 62.6 1000 64.3 FIG> 4B TSD1238S205′CCGCCCTTCTCTCTGCCCCCTGGT3′ 8 300 71.2 10 66.7 +2.4 TSD1301A225′-GCCAGGGGTTCCACTACGTAGA-3′ 2 300 62.6 1000 64.3

[0096] FIGS. 5A-5C illustrate that well-designed LATE-PRC primersgenerate efficient reactions over a wide range of Limiting Primer toExcess Primer ratios. In this case, the HEX-A primers used in FIG. 4Bwere prepared at three different ratios: 1:10, 1:40, 1:100. The startingconcentration of the Excess Primer was held constant (at 1000 nM) ineach case, while the starting concentration of the Limiting Primerdecreased (from 100 nM to 25 nM to 10 nM). The efficiencies and kineticsof these assays (curves 51, 53, and 55, respectively) were compared toan assay containing equimolar concentrations of the same two primers(500 nM:500 nM, curves 52, 54, and 56). Each assay was performed inreplicate (5 reactions each) and the averages of these replicates areshown in FIGS. 5A-5C. The results show that all three LATE-PCR reactions(curves 51, 53, and 55) were efficient, i.e. had C_(T) values equivalentor slightly lower than the symmetric PCR assay (curves 52, 54, 56), andthey did not plateau while the symmetric reaction did. Analysis of theprimer sets used in FIG. 5 is provided in Table III and explains theseresults in terms of the principles of LATE-PCR. All three asymmetricreactions in FIG. 5 (curves 51, 53, and 55) have (T_(m[0]) ^(L)−T_(m[0])^(X))≧0. TABLE III Comparison of (T_(m[0]) ^(L)-T_(m[0]) ^(X)) fordifferent ratios of primers Primer Primer Conc. Primer Set Ration (nM)T_(m[0]) (° C.) (T_(m[0]) ^(L)-T_(m[0]) ^(X)) TSD1238S20 1:10 100 69.8+5.5 TSD1301A22 1000 64.3 1:40 25 67.9 +3.6 1000 64.3 1:100  10 66.7+2.4 1000 64.3

[0097] Designing the Excess Primer in Relation to the Amplicon

[0098] LATE-PCR, in contrast to most conventional symmetric andasymmetric PCR, also takes the melting point of the amplicon, T_(m)^(A), into account, particularly as it relates to theconcentration-adjusted melting point of the Excess Primer. Becauseamplicons are almost always greater than 50 nucleotides long, wecalculate the T_(m) ^(A) of the amplicon by the “% GC” method, seeabove. While other mathematical formulas or even other means, such astrial and error, can be used for design of primers according to thisinvention, melting-point relationships described herein are analyzedusing the formulas given above. These formulas are useful both fordesign and evaluation despite the fact that they do not consider theconcentration of magnesium ion (Mg⁺⁺), which is almost universallyincluded in PCR reaction mixtures in concentrations from 1 to 5 mM, andaffects the melting points of primers and amplicons. Example 5 describesour design of different primer pairs taking T_(m) ^(A) into account, aswell as certain primer properties that we prefer.

[0099] For any chosen DNA target (for instance the sequence flanking amutation in a particular gene) T_(m) ^(A) is usually similar forcandidate pairs of Limiting and Excess Primer. For this reason theapproximate size of the amplicon (and approximate location of theprimers) is chosen first, establishing the approximate T_(m) ^(A).Several possible Excess Primers are selected next, such that (T_(m)^(A)−T_(m[0]) ^(X)) is within the preferred range of 7-18° C., morepreferably 7-15° C., for amplifications, or 7-25° C., more preferably7-18° C. and most preferably 7-18° C., for real-time assays. Once a setof possible Excess Primers has been selected, the sequence of the targetis examined for the presence of possible GC-rich sequences at which todesign candidate Limiting Primers. Several possible Limiting Primers arenext designed for a range of possible Excess/Limiting primer ratios,with the goal of making sure that (T_(m[0]) ^(L)−T_(m[0]) ^(X))≧0.

[0100]FIG. 6 shows a set of such reactions in which (T_(m[0])^(A)−T_(m[0]) ^(X)) is varied from +7 to +19° C., and (T_(m[0])^(L)−T_(m[0]) ^(X)) is specifically set to zero. The experimentaldetails for these data are described in Example 5. Each curve representsthe average increase in molecular beacon fluorescence of 3 replicatesamples. Assays with (T_(m) ^(A)−T_(m[0]) ^(X))=12 (curve 61) yieldedthe strongest beacon signal (i.e., the largest quantity ofsingle-stranded CFTR product) in this series. Samples with (T_(m)^(A)−T_(m[0]) ^(X))=19 (curve 65) yielded the lowest signal. Sampleswith intermediate values of (T_(m[0]) ^(A)−T_(m[0]) ^(X))=14 (curve 62),or 16 (curve 64) yielded intermediate average signal intensitycorresponding with that value. Samples with (T_(m) ^(A)−T_(m[0]) ^(X))=7(curve 63) also yielded intermediate final signal intensity.

[0101]FIG. 7 illustrates several real-time reactions in which (T_(m)^(A)−T_(m[0]) ^(X)) varies from +13 to +23° C. and (T_([0])^(L)−T_(m[0]) ^(X))=5-to-6° C. in all cases, (Example 5 Table IX). Thehighest mean molecular beacon signal (cycles 35-60) were in samples with(T_(m) ^(A)−T_(m[0]) ^(X))=13 (curve 71), indicating efficient singlestrand synthesis. The mean intensity of the molecular beacon signaldecreased with each increase in (T_(m) ^(A)−T_(m[0]) ^(X)) to values of17 (curve 72), 19 (curve 73), 20 (curve 74), and 23 (curve 75). None ofthese samples showed an amplification plateau, illustrating anotheradvantage of having (T_(m[0]) ^(L)−T_(m[0]) ^(X))≧5° C. Electrophoresisof these samples revealed only the specific single- and double-strandedamplicon. Experimental details of the data in FIG. 7 are provided inExample 5.

[0102] Primers for amplifications and assays according to this inventionmay utilize universal primer sequences added to the 5′ end of one orboth primers of a pair. Universal priming sequences have particularutilities, such as introduction of specific restriction enzymesequences, or to enhance multiplexing. A universal priming sequenceincluded in a Limiting Primer will not raise its melting temperaturewith the intended target during the initial cycles of amplification,when specificity is particularly crucial, but it will raise its meltingtemperature thereafter due to the generation of amplicons containingsequences complementary to the universal priming sequence. While theannealing temperature during the first few, generally 5-10, cycles maybe lowered to improve efficiency when the Limiting Primer contains auniversal sequence addition, care must be taken not to incurnon-specific amplification due to non-specific hybridization of theExcess Primer that may result. In some instances it may be preferable tosacrifice efficiency during the first few cycles by using a higherannealing temperature according to the T_(m) of the portion of theprimer that is complementary to the starting targets. If a universalpriming sequence is added to the Excess Primer, there will be a similarmelting point increase after the first few cycles, which will reduce themelting-point difference between the two primers. If a universal primingsequence is added to the Limiting Primer, or any other mismatch isintroduced into the Limiting Primer, such that the Limiting Primer doesnot perfectly hybridize along its entire length to its initial target,the concentration-adjusted melting point of the entire primer, T_(m[0])^(L), is designed to be greater than or equal to theconcentration-adjusted melting point of the Excess Primer T_(m[0]) ^(X),with the added proviso that the functional concentration-adjustedmelting point of the portion of the Limiting Primer to its initialtarget sequence is not more than 5° C. lower than, and preferably atleast equal to, the concentration-adjusted melting point of the ExcessPrimer. Primers useful in this invention may contain modifiednucleotides, by which we mean generally nucleotides different from thefour natural dNTPs. Such nucleotides may affect primer melting point. Ifa mathematical formula cannot be located for the effect of a particularmodified nucleotide, the concentration-adjusted melting point, T_(m[0]),can be determined empirically.

[0103] Protocol for Optimizing the Absolute Concentration of theLimiting Primer and Excess Primer.

[0104] In real-time LATE-PCR assays it is desirable for the LimitingPrimer to be depleted at about the same thermal cycle that thedouble-stranded product of the reaction first becomes detectable abovebackground, the C_(T) value of the reaction. As is known in the art, thecycle at which the C_(T) value is reached depends, among other things,on the amount of the target DNA present at the start of the reaction,the efficiency of amplification, the nature of the detection equipment,and the intensity of the signal (usually a fluorescent or electricalsignal) generated by the hybridization probe. In LATE-PCR one of theprimers is depleted after about 15-35 PCR cycles, after which linearamplification of one strand takes place during subsequent cyclesutilizing the Excess Primer. In order to maximize the amount of singlestranded product it is useful to optimize the absolute amount of theLimiting Primer for any chosen ratio of primers. In practice, it is alsodesirable to avoid Limiting Primer concentrations that exceed 200 nM.Above this concentration, the prolonged exponential phase of thereaction may produce a ratio of double-stranded to single-strandedproduct that is unacceptable for some applications, and may actuallyreduce the total amount of single-stranded product generated. Also, at aratio of 10:1 or higher the Excess Primer concentration would be pushedabove 2000 nM. Under these conditions it is difficult to avoidnon-specific initiation of amplification.

[0105] The preferred concentration of Limiting Primer depends mainly onthe general nature of the probe (e.g., the intensity of fluorescencefrom the hybridized vs. the unhybridized state), the sensitivity of thedetection equipment, and the ability of the specific probe to hybridizeto its target at the detection temperature. The Limiting Primerconcentration needed is less dependent on the initial targetconcentration, since the increased target numbers will simply exhaustthe Limiting Primer at an earlier thermal cycle, at which the probesignal becomes detectable.

[0106] One method for choosing the concentration of Limiting Primer thatyields the desired transition from exponential to linear amplificationis through empirical determination. First, several LimitingPrimer/Excess Primer pairs are designed for testing one or more primerratios (e.g., 1 to 20). Next, each of the primer pairs is empiricallytested at several annealing temperatures, annealing times, and/ormagnesium concentrations to determine which pair of primers and whichconditions generate the intended specific amplicon with highestefficiency and specificity. In real-time reactions overall amplificationefficiency can be followed by use of SYBR® Green to determine the C_(T)value of the reaction. Optimal annealing conditions must be determinedfor each concentration of primer pairs that are to be assayed using asequence-specific probe, see below.

[0107] Next, amplification is carried out in the presence of thespecific probe under optimal conditions for several concentrations ofthe Limiting Primer in the range expected for the probe that will beused. When the probe is a molecular beacon, the preferred LimitingPrimer concentration is found in the range of 10 nM to 100 nM. Thepreferred Limiting Primer concentration corresponds to the lowestLimiting Primer concentration that yields a mean C_(T) value similar tothose from samples with higher concentrations of that primer.Concentration below the preferred concentrations show increases of morethan about 1 cycle as the concentration is decreased, which indicatesthat, under this circumstance, Limiting Primer depletion occurs too farin advance of reaching the detection threshold. In addition, sampleswith the preferred Limiting Primer concentrations will show linear rateof signal increase for several cycles after reaching threshold withoutplateauing, usually even 30 cycles after initial detection.

[0108] Protocol for Optimizing the Annealing Temperature for LATE-PCR

[0109] Once a suitable Limiting/Excess Primer pair has been chosen foruse at a fixed ratio and absolute concentration, the T_(m[0]) of eachprimer will have been, or can be, calculated using the Nearest Neighborformula as described above. This done, it is important to empiricallyestablish the Optimal Annealing Temperature at which to use theseprimers. The Optimal Annealing Temperature is the highest temperature atwhich the exponential phase of the reaction proceeds with maximalefficiency and maximal specificity at specific reagent concentrationsand cycling times. By “maximum efficiency” we mean the condition thatgenerates the lowest C_(T) value during the exponential phase ofreaction, wherein the specific product accumulates at the highest rate.As the annealing temperature is adjusted further downward toward theOptimal Annealing Temperature, the efficiency of the exponential phaseof the LATE-PCR tends to increase. As the annealing temperature isadjusted downward below the Optimal Annealing Temperature, reactionstend to amplify non-specific amplicons. If, as is sometimes the case,the pair of primers being used does not hybridize non-specifically toalternate sequences within the target sample, decreasing the annealingtemperature below the Optimal Annealing Temperature does not increasethe efficiency of the reaction significantly and does not substantiallydecrease reaction specificity. Thus, the term Optimal AnnealingTemperature as used in this application has a specificempirically-defined value, even for amplifications where lowering theannealing temperature below that temperature is not detrimental.

[0110] Protocol for Product Detection in Conventional Real-Time PCR

[0111] In the case of conventional real-time PCR the double-strandedproduct is detected by inclusion of a fluorescent dye or some type of alabeled probe, typically a fluorescent probe. Detection of doublestrands using a fluorescent dye may be done during the step of primerextension. The hybridization probes typically bind to one or bothstrands of the amplicon as the reaction is being cooled between thesteps of strand melting and primer annealing. In practice this meansthat the melting temperature of the probe is higher than the meltingtemperature of the primer which hybridizes to the same strand as theprobe (Mackay, I. M. (2002) “Survey and Summary: Real-time PCR inVirology”, Nucleic Acids Res. 30(6):1292-1305). As the reaction iswarmed again the probe is designed to disengage from the target strandwhile the primer extends along the target strand. Hybridization andextension of the other primer on the complementary strand also takesplace during these steps. Probes that generate the detected signalthrough hybridization are detected during the annealing step. Probesthat generate the detected signal through being hydrolyzed are detectedfollowing their degradation during the subsequent extension step. Ineither case, the amount of hybridized probe is limited by reannealing ofthe strands of the amplicon as their concentration increases. This meansthat only a fraction of the total number of target sequences present atthe end of a conventional real-time reaction are actually detected.

[0112] In addition, under conventional real-time reaction conditions thehybridization probe must either fall off the target sequence prior tothe extension step or be hydrolyzed during the extension step. If thehybridization probe fails to melt off its template strand quickly as thetemperature is raised from primer annealing to primer extension in asymmetric PCR reaction, we have found that the probe may interfere withprimer extension and reduce the amplification efficiency of thereaction.

[0113] Protocol for Product Detection in Real-Time LATE-PCR

[0114] Real-time LATE-PCR assays, like real-time symmetric PCR assays,include one or more labeled probes or fluorescent dyes for detection ofthe double-stranded and/or single stranded products. Again as in thecase of symmetric PCR, detection of double-stranded product synthesizedduring the exponential phase of LATE-PCR can be carried out duringeither the primer-annealing or the primer-extension step, mostpreferably during the primer-extension step. The double-stranded productgenerated during the exponential phase of the LATE-PCR can be detectedusing a dye that binds double-stranded DNA, such as SYBR® Green.However, double-stranded DNA cannot be detected during a low-temperaturedetection step by use of a hybridization probe, such as asingle-stranded probe or a molecular beacon, because the vast majorityof the two amplicon strands are re-annealed to each other at the lowtemperature.

[0115] In one embodiment of the invention, detection of the accumulatingsingle-stranded molecules can be carried out during the annealing step,i.e. prior to the extension step. It is preferred that the probe be aLow-T_(m) Probe with a T_(m[0]) ^(P) at least 5° C., preferably at least10° C. below T_(m[0]) ^(L). (Dual-labeled linear probes for the 5′nuclease assay (for example TaqMan® probes) are never Low-T_(m) Probes,because they must remain bound to the template strand until they aredegraded during primer extension.) Under these conditions Low-T_(m)Probes detect the accumulating single-strands plus a fraction of thetarget strands that would otherwise reanneal to their complementarystrands if the probe were not present. The exact magnitude of thefraction depends on the T_(m[0]) ^(P) as well as the reaction conditionsand tends to vary slightly among replicate reactions, therebyintroducing a variable in these measurements. In order to minimize thiserror it is preferred, for detection during the annealing step, to use aSuper-Low-T_(m) probe and to reduce the temperature of the annealingstep below the mean annealing temperature for primer hybridizationduring the exponential phase of the reaction, with the proviso that thischange in the thermal profile should not lead to mis-priming. Ourpreferred method to avoid mis-priming under these circumstances is toonly lower the annealing temperature at, or preferably a few cyclesbefore, the cycle at which the Limiting Primer becomes depleted and thereaction switches to synthesis of the Excess-Primer-Strand only.

[0116] In a more preferred embodiment of the invention, a detection stepis introduced into the thermal profile of the reaction between primerextension of one thermal cycle and strand-melting of the next thermalcycle. In this case, the detection step is introduced for the purpose ofdetecting accumulating single-strand molecules after the extension stepis finished, using hybridization probes that are complementary tosequences within the strand formed by extension of the Excess Primer.Detection can be carried out at any temperature at which the probehybridizes to its target, in most cases below the extension temperature,and preferably at or below the annealing temperature, in combinationwith a Low-T_(m) Probe. In the most preferred embodiment of theinvention the added detection is carried out at a low temperature,preferably 5° C. below and most preferably 10° C. below, the meantemperature of the annealing step of the exponential phase of thereaction and utilizes a Super-Low-T_(m) probe. Detection of thesingle-stranded product is most accurate when detection is carried outafter the extension step of one cycle and the melting step of the nextcycle, as compared to detection during the annealing step.

[0117] Introduction of a separate detection step into LATE-PCR,preferably a low-temperature detection step, has several advantages overthe conventional strategy of probe detection prior to/or during theextension step. It makes it possible to separate primer annealing andextension from probe hybridization and detection. This, in turn, makesit possible to use elevated annealing temperatures and/or extremelyshort annealing times (such as “touch-down” annealing), designed toincrease the stringency of the reaction and decrease the chances ofamplifying an incorrect amplicon. It also makes it possible to routinelyutilize non-hydrolysable Low-T_(m) and/or Super-Low-T_(m) probes insteadof conventional hybridization probes. Also introduction of alow-temperature detection step also makes it possible to monitor thepresence of the extension product of the Limiting Primer (theLimiting-Primer Strand) that remains constant during the linear phase ofLATE-PCR, while simultaneously measuring the accumulation of theExcess-Primer-Strand that increases linearly during the linear phase ofLATE-PCR. This can be done by use of a labeled Limiting Primer that isincorporated into the Limiting-Primer-Strand, and also using a Low-T_(m)Probe or a Super-Low-T_(m) Probe that gives off its own distinct signalto measure the accumulating Excess Primer-Strands. In addition, becausethe temperature of the reaction is immediately increased to the meltingtemperature when the detection step is used, there is a decreasedopportunity for a mis-match primer to extend. In contrast, when aconventional probe is used, a mismatched primer has an excellent chanceof extending, because the conventional detection step during primerannealing is followed by the conventional extension step.

[0118] Advantages of Low-T_(m) Probes and Super-Low-T_(m) Probes

[0119] Low-T_(m) and Super-Low-T_(m) probes have the followingadvantages as compared to conventional hybridization probes: a) theseprobes increase signal strength because, when used at highconcentrations, they bind to all accumulated single-strands; b) theseprobes are more allele specific than conventional probes; and c) theseprobes have a better signal-to-noise ratio than conventional probes,because they are detected at lower temperatures at which non-specificfluorescence background is typically lower.

[0120] Protocol for Designing and Characterizing Low-T_(m) Probes

[0121] Low-T_(m) probes and Super-Low-T_(m) probes compatible withLATE-PCR include, but are not limited to, one or more probes of thefollowing types: 1) single-stranded linear probes labeled as is known inthe art, including FRET probes; 2) double-stranded probes, as is knownin the art; 3) stem-loop single-stranded probes, including molecularbeacons, labeled as is known in the art. These general classes includeprobes containing unconventional nucleotides such as PNA or 2-O-methylmodifications, probes with attached moieties that affect hybridstability such as Minor-Grove-Binding probes (e.g., Eclipse™ probes),and probes that are physically attached to primers (e.g., Scorpionprobes). Low-T_(m) probes and Super-Low-T_(m) probes are constructedbased on the following logical steps:

[0122] 1) The experimenter chooses which type of probe is to be used;

[0123] 2) The experimenter decides on the probe's target sequence,approximate length, and chemical composition. The experimenter designsthe probe using an appropriate software package, while applying certainwell known general principles of nucleic acid biochemistry. Manysoftware packages for this purpose are known in the art, but are notavailable for types of probes comprised of non-conventional subunits.The software package may offer an approximate estimate of T_(m[0]) ^(P)of the probe. The following general principles are useful for designingthese probes: a) under constant experimental conditions shorterprobe/target hybrids tend to have lower T_(m) values than longer probes;b) under constant experimental conditions probes/target hybrids withfewer hydrogen bonds tend to have lower T_(m) values than probes withmore hydrogen bonds; c) under constant experimental conditionsprobe/target hybrids that are mismatched tend to have lower T_(m) valuesthan probe/target hybrids that are perfectly matched.

[0124]3) The experimenter empirically establishes the approximateT_(m[0]) ^(P) of one or more possible probes using a melting temperatureassay. Many types of melting temperature assays for probe/target hybridsare well known in the art. One version of a melting temperature assay isoutlined here. An experimental mixture of the probe and its target isprepared under conditions that simulate the composition of the LATE-PCRreaction mixture. Tests are prepared at probe:target ratios of 1:1 andone or more probe concentrations within the sensitivity range of theinstrument that will employed for the melting temperature assay. Thesemixtures are then subjected to a melt temperature assay. In the case offluorescently labeled probes the melting temperature assay is carriedout in a fluorimeter having thermal regulation. The melting temperatureof each probe-target pair is the temperature at which 50% of the probemolecules are hybridized to the target molecules. The experimentallydetermined melting temperature is the T_(m[0]) ^(P) of that probe underthose conditions. However, as will be understood by those skill in theart, this empirically determined value is different from the actualmelting point of probe-target hybrids in an actual LATE-PCR. This isbecause the concentration of the target added to melting point assay isequal to the concentration of the probe, whereas in a LATE-PCR the probeis in excess of the concentration of the target, which begins thereaction at zero and almost never reaches the probe concentration overthe course of the reaction.

[0125] 4) In the case of Low-T_(m) Probes, the empirically establishedT_(m[0]) ^(P) melting temperature should be at least 5° C. below, mostpreferably at least 10° C. below, T_(m[0]) ^(L) of the Limiting Primer.In the case of Super-Low-T_(m) Probes, the empirically establishedT_(m[0]) ^(P) should be below, preferably at least 5° C. below, morepreferably at least 10° C. below, the mean annealing temperature usedfor the primer pair during the exponential phase of the reaction.

[0126] 5) The low-temperature detection step, when used, does not needto be included in every thermal cycle of LATE-PCR. In fact, undercertain circumstances it is desirable to use a Low-T_(m) probe orSuper-Low-T_(m) probe without including the detection step until the endof the reaction. For instance, it may be desirable to keep thestringency of the single-strand amplification phase very high for manycycles to prevent Product Evolution (discussed below) or mis-priming.Under these circumstances dropping the temperature below that requiredfor stringency, during any step in the thermal cycle, could encouragethe Product Evolution of single strands, or could lead to generation ofnon-specific products. However, once the desired number ofsingle-stranded product molecules has accumulated in LATE-PCR,introduction of a low temperature detection step can be used to measurethe amount of the accumulated single strands. If SYBR® Green is alsopresent in the reaction, or a second probe to the opposite strand isalso present in the reaction, a measure of the number of double strandedmolecules can also be obtained. The resulting data, together withknowledge of the thermal cycle at which the LATE-PCR switched fromexponential amplification to linear amplification, can be used toestimate the efficiency of single-strand synthesis in the reaction.

[0127] Protocol for Optimizing Low-T_(m) Probe and Super-Low-T_(m) ProbeConcentrations for LATE-PCR

[0128] Replicate LATE-PCR assays are performed each with an initialprobe concentration ranging above or below that used to establish theempirical T_(m[0]) ^(P) for that probe (see above). The intensities ofthe signals generated in reactions are compared and the optimal probeconcentration is chosen as the lowest concentration that gives themaximum signal. For instance, FIG. 8 shows four parallel LATE-PCR assayseach containing a different concentration of a Low-T_(m) molecularbeacon (0.6 μM, curve 81; 1.2 μM, curve 82; 2.4 μM, curve 83; 4.0 μM,curve 84). The results show that 2.4 μM (curve 83) is the optimalconcentration for this Low-T_(m) Probe under the conditions of thisLATE-PCR.

[0129] Protocol for Optimizing the Absolute Concentration of theMagnesium in a LATE-PCR

[0130] Alterations in Mg⁺⁺ concentration affect many other aspects ofthe LATE-PCR, as is the case in conventional PCR. The factors affectedinclude the T_(m) ^(A), T_(m[0]) ^(L), T_(m[0]) ^(X), the optimalannealing temperature, T_(m[0]) ^(P), closing of the stem of a molecularbeacon, and the activity of Taq DNA polymerase. Generally, the T_(m) ofeach component in the reaction increases as the Mg⁺⁺ concentrationincreases, but the specificity of interactions between eacholigonucleotide and its target sequence decreases. These effects of Mg⁺⁺are well known to persons skilled in the art of PCR. It is thereforenecessary to empirically define the optimal Mg⁺⁺ through a series ofparallel reactions. We prefer optimal Mg⁺⁺ concentrations in the rangeof 1-6 mM, most preferably in the range of 2-4 mM.

[0131] LATE-PCR Assay Kits

[0132] A LATE-PCR reagent kit has been designed for use in the detectionof the normal and ΔF508 Alleles of the human cystic fibrosis gene duringpreimplantation genetic diagnosis (PGD). The kit described here ismodular; that is, it contains DNA polymerase in one package and allother reagents and materials in another package. It will be appreciatedthat the primers and probes together comprise an oligonucleotide set,which can be marketed as a separate product. The kit, its use, and theassay performed with the kit, which we call the “CFΔ508 Kit,” aredescribed in Example 6 in a format that might appear on a product insertaccompanying the kit.

[0133] Similar kits can be designed for use with other targetsgenerally, including but not limited to single cells from sources otherthan human embryos, or pluralities of cells, or DNA or RNA recoveredfrom plant or animal cells or other sources cells. In the case ofsamples comprised of RNA the LATE-PCR kit can be used in conjunctionwith a variety of procedures known in the art for isolation orpurification of RNA and conversion of said RNA into cDNA.

[0134] LATE-PCR Assays Based on the Use of Three Primers

[0135] Certain embodiments of LATE-PCR assays use an additional LimitingPrimer to generate a relatively long double-stranded amplicon from twoLimiting Primers during the initial, exponential phase of the reaction,followed by generation of a shorter single-stranded amplicon utilizingone strand of the long amplicon as template and the Excess Primer as theprimer. While this can be accomplished by opening the reaction vessel toadd the Excess Primer after multiple cycles of exponentialamplification, preferred LATE-PCR 3-primer assays utilize initialreaction mixtures that contain all three primers together with anamplification protocol that preferentially utilizes the Excess Primeronly in later cycles. The Limiting Primers are a pair of matched PCRprimers, designated L1 and L2, for generating the large amplicon. Theprimers are “matched”; that is their T_(m)'s are “balanced”. Theconcentration of L2 is roughly equal to the concentration of L1, thatis, from 0.2-5 times the concentration of L1, preferably equimolar. Theinitial concentration-dependent melting temperatures, T_(m[0]) ^(L1) andT_(m[0]) ^(L2), of L1 and L2 are as close as possible to one another,while T_(m[0]) ^(X), of the Excess Primer is at least 5° C., preferablyat least 10° C., below the T_(m[0]) of both Limiting Primers. Initialcycles of PCR amplification, either 3-step PCR or 2-step PCR, utilize anannealing temperature higher than the T_(m[0]) ^(X) of the ExcessPrimer, such that the Excess Primer does not participate materially inthe generation of amplicons. After a selected number of thesehigh-temperature cycles, preferably near the point exponentialamplification ceases due to depletion of L1 and L2, the annealingtemperature is lowered such that during subsequent cycles the ExcessPrimer participates in amplification (exponential amplification, if itspairing Limiting Primer has not been completely exhausted, followed bylinear amplification of single-stranded LATE-PCR product). Thus, therelationship between the melting temperature of the shorter ampliconformed by extension of the Excess Primer, that is T_(m) ^(A) of theamplicon is not more than 25° C. above T_(m[0]) ^(X) of the ExcessPrimer, preferably not more than 20° C. above and more preferably notmore than 18° C. above.

[0136] The present invention is an improvement in the digital PCR method(Vogelstein, B., & Kinzler, K. W. (1999) “Digital PCR” Proc. Natl. Acad.Sci. USA 96:9236-9241). According to this method single DNA moleculesare amplified by symmetric PCR. Once the symmetric reaction is completeda single additional primer is added to the reaction to amplify just onestrand of the accumulated double-stranded molecules. The resultingsingle strands are then detected by addition of an appropriatefluorescent probe, or by electrophoresis. LATE-PCR can be used to carryamplification of both the double-stranded product and the singlestranded product in one reaction. In some applications, such as thedetection of cancer DNA molecules present in feces (Shih, I., et al.(2001) “Evidence That Genetic Instability Occurs at an Early Stage ofColorectal Tumorigenesis” Cancer Res. 61:818-822), LATE-PCRamplifications can be carried out in situ, such as in agarose orpolyacrylamide gel.

[0137] Quantitative LATE-PCR Assays

[0138] Assays based on LATE-PCR allow quantitative measurement to beobtained in three ways. First, real-time LATE-PCR can be used to measurethe C_(T) value of a signal. As in the case of real-time symmetric PCR,the C_(T) value can be used to deduce the number of target moleculespresent in the initial sample. This is accomplished by comparing theC_(T) value of the sample with a standard curve generated by analyzingknown amounts of the same target sequence under conditions that simulatethose of the unknown sample. Second, the slopes of the signal during thelinear amplification phase of the real-time LATE-PCR can be measured. Asillustrated below for a single copy sequence, the linear slopes ofhomozygous diploid cells are approximately twice those for the samesequence in heterozygous diploid cells. Third, LATE-PCR under optimizedconditions can be used to determine the relative number of differentallelic copies present by means of end-point assays.

[0139] End-point LATE-PCR assays can also be used to provide anestimation of the number of targets in the original sample, if therelative number of alleles is known and the slope of the line is similaramong replicate samples. This is possible because LATE-PCR reactions donot plateau but continue to increase linearly for many cycles. Thus,once the expected C_(T) values of such reactions have been established,single data points can be used to extrapolate the slopes of the linesand hence the number of target molecules present at the start of thereaction. Similarly, if the number of target molecules and expectedC_(T) values are first established, end-point assays can be used toquantify the frequencies of different allelic sequences among them.

[0140] In the case of both real-time and end-point assays, it will beappreciated that both the double-stranded products and thesingle-stranded products of a LATE-PCR amplification can be monitoredsimultaneously by use of a combination of dyes and hybridization probes,or a combination of hybridization probes and primer probes. Listed beloware some possible strategies that can be used but, as will beappreciated by those skilled in the art, additional strategies arepossible:

[0141] In the case of single amplicons two hybridization probes can beused to simultaneously measure the synthesis and accumulation of theextension product of the Limiting Primer that stops being synthesized atthe end of the exponential phase, as well as the extension product ofthe Excess Primer that continues to accumulate linearly.

[0142] Alternatively, accumulation of the extension product of theLimiting Primer can be monitored using a labeled double-stranded primerand its quenched complementary strand (as described by Li et al., whilethe extension product of the Excess Primer can be monitored by use of anappropriate hybridization probe, such as a molecular beacon or adouble-stranded probe.

[0143] Alternatively, accumulating double-stranded amplicons can bemonitored by binding an intercalating dye, such as SYBR Green®, whilethe single-stranded extension product of the Excess Primer can bemonitored by use of an appropriate hybridization probe, such as amolecular beacon.

[0144] In the case of multiplex reactions several probes can be used tosimultaneously monitor several strands that continue to accumulateduring the single-strand phase of the reaction.

[0145] LATE-PCR Assays Used to Establish Genomic Zygosity

[0146] Assays of this invention permit the discrimination betweengenomes that are homozygous for a particular allele versus those thatare heterozygous for that allele. Assays that can distinguish betweenhomozygous cells and heterozygous cells can be performed starting withsingle cells or single genomes, but other samples may also be used,provided that the two samples being compared have approximately the sameamounts of DNA at the start of the reaction. Assays that can distinguishbetween homozygous and heterozygous cells are clinically or commerciallyimportant, because they can distinguish between organisms that do or donot carry one or two copies of a particular allele or a variant of thatallele.

[0147] As shown in FIG. 9, the presence of one copy of a selectednucleic acid sequence present in a heterozygous diploid cell can bedistinguished from the presence of two copies of the same nucleic acidsequence in a homozygous diploid cell by means of real-time LATE-PCRassay in which one molecular beacon is used to detect one of the allelesin the heterozygous cell, or two differently-colored molecular beaconsare used, one for one allele and the other for the other allele in theheterozygous cell. The resulting fluorescent signals generated from suchsamples demonstrate that, for each allele, the linear slope of thesignal arising from homozygous cells containing two copies of thatparticular allele (curve 91) increases at a rate that is approximatelytwice the rate of the signal for the same allele generated by theequivalent number of heterozygous cells that contains one copy each oftwo different alleles (curve 92)

[0148] It will be appreciated that for this use of the invention it isparticularly important that the slopes of the lines generated during thesingle-strand phase of the reaction be optimized for reproducibility,that is, show the least possible scatter among replicates. In thisregard it is most preferred that (T_(m[0]) ^(L)−T_(m[0]) ^(X))≧+3. FIG.9 illustrates such an optimized case for genomes that are homozygous vs.heterozygous for the 1421 allele of the HEX-A gene, one of the commonalleles responsible for Tay-Sachs Disease. Each of curves 91, 92 in FIG.9 is the average of 15 replicate tests. It is apparent in this examplethat the homozygous normal DNA (assayed for the wildtype allele) has anaverage slope approximately twice as steep as the average slope for thewildtype allele present in cells heterozygous for the 1421 allele.Either the slopes of the curves generated by LATE-PCR assays, orend-point values generated by LATE-PCR assays can each be used todistinguish between homozygous and heterozygous cells.

[0149] LATE-PCR also can be used to distinguish between genomes that areheterozygous for a particular allele and genomes that are hemizygous forthe same allele. Assays that can distinguish between hemizygous cellsand heterozygous cells can be performed starting with single cells orsingle genomes, but other samples may also be used, provided that thetwo samples being compared have approximately the same amounts of DNA atthe start of the reaction. Assays that can distinguish betweenhemizygous and heterozygous cells are clinically or commerciallyimportant because, among other phenomenon, they can be used to detect“loss of heterozygosity” a well known event that takes place in certaincancers and in normal cells of the immune system undergoingrecombination and loss of a portion of the immunoglobin genes during thecourse of cellular differentiation. In such cases a small piece or largepiece of one chromosome is lost, thereby rendering a portion of thegenome hemizygous. Heterozygous cells generate signals in a LATE-PCRassay, monitored with an appropriate probe, that have slopes and/orend-points that are approximately one-half those generated by the sameprobe monitoring amplification of the DNA from a hemizygous cell.

[0150] Multiplex LATE-PCR Assays

[0151] Assays according to this invention include multiplex assays forsimultaneous amplification of two or more target sequences withdifferent primer pairs in the same reaction mixture. For multiplexassays, it is recommended that the various primers be analyzed to screenout obvious cases of undesirable cross-hybridization between twoamplicons and between one primer pair and amplicons from other primerpairs. For multiplex assays, the concentration-adjusted meltingtemperatures, T_(m[0]) ^(L), of all Limiting Primers should be equal toor higher than the concentration-adjusted melting temperatures, T_(m[0])^(X), of all Excess Primers. Preferably, the linear phase of multiplexamplifications is carried out under stringent conditions to minimizefalse priming. FIG. 10 shows the results of a multiplex assay accordingto this invention. Two separate amplicons were synthesized from theHEX-A gene. One target sequence included the site for the 1278 mutation.The other target sequence included the site for the 1421 mutation.Differently labeled (TET in one case, FAM in the other) molecular beaconprobes were used to monitor the two amplicons in real time. The twoplots of fluorescence in FIG. 10 (curves 101 and curve 102) show thatboth targets were amplified successfully.

[0152] Assays according to this invention, particularly multiplexassays, may include the use of universal priming sequences. We havedesigned a multiplex assay that uses a primer pair for each amplicon inwhich each Limiting Primer has a universal 5′ sequence and that alsouses an extra primer, a universal primer, that includes only theuniversal sequence. The universal primer has a concentration-adjustedmelting temperature, T_(m[0]) ^(U), that is lower than T_(m[0]) ^(X) ofthe Excess Primers. Limiting Primers and Excess Primers haveconcentration-adjusted melting points as described above. They are addedat the described ratio, for example, 1:20 or greater, but at very lowconcentration, for example 1 nM for the Limiting Primers. An example ofpreferred concentrations is I nM for the Limiting Primers and 50 nM forthe Excess Primer. Initial cycles of amplification are at conditionsappropriate for the primer pairs, and the Limiting Primers are exhaustedafter a relatively few cycles. The universal primer does not participatein these initial cycles. From about that point onward the Excess Primersfunction as “Limiting Primers” relative to the universal primer, whichhas a concentration-adjusted melting temperature, T_(m[0]) ^(U), lowerthan those of the Excess Primers present in the reaction, but is presentat a concentration at least 5 times, preferably at least 10 times,greater than the concentration used for the Excess Primers. Furthertemperature cycles during this second phase of the amplification utilizea lowered annealing temperature appropriate for the universal primer.After exhaustion of the Excess Primers, continued cycling leads tosynthesis of single-stranded products via extension of the universalprimer. In essence, this method couples a first LATE-PCR amplificationto a second LATE-PCR reaction, wherein the Excess Primer(s) in the firstamplification are the Limiting Primer(s) in the second.

[0153] The efficiency of the multiplex assay described above may belimited during the initial cycles of the reaction because theconcentrations of the Limiting Primers and the Excess Primers are low.If necessary, the initial efficiencies of amplicon production can beincreased by raising the concentrations of these primer pairs. Raisingthe concentration of these primer pairs can be accomplished by a volumechange, that is, by carrying out the first phase of the amplificationutilizing a much smaller reaction mixture volume than the reactionmixture volume of the second phase. Under these conditions the volume ofthe reaction can be increased at or near the thermal cycle at which thetemperature of the annealing phase is lowered to allow the universalprimer to begin functioning. An alternate version of the multiplexassays described above is to add the universal primer to the assay atthe time it is first needed.

[0154] Additional LATE-PCR Amplifications and Assays.

[0155] It may be desirable to convert one or more of the single-strandproducts in a LATE-PCR amplification back into double-stranded products.This can be accomplished by including a “Low-T_(m) Primer” in areaction. A “Low-T_(m) Primer” only hybridizes to its complementarysequence when the temperature is dropped below the T_(m[0]) value ofsaid primer during an additional step included in thermal cycles late inthe reaction and then is slowly raised to allow for extension of saidprimer and, hence, allowing the accumulated single-stranded molecules tobe converted back into double-stranded DNA. If initial attempts atconversion prove inefficient, the time spent at the low temperature stepcan be lowered and/or the rate of temperature increase from the lowtemperature can be slowed. Alternatively several down-and-up temperatureoscillations, for instance between 45° C.-72° C.-45° C.-72° C.— . . .can be carried out prior to either ending the reaction or continuing onto the melting step of the next thermal cycle. A version of thisembodiment of the invention is to design a “Low-T_(m) Primer” that canhybrid to a sequence within the accumulating single-stranded product,rather than at or near its 5′ end. In this case, only the portion of thesingle-strand that is 3′ of the “Low-T_(m) Primer” is converted to adouble-strand. The product strands of this reaction can becomesubstrates for synthesis of additional truncated single-strands usingthe original Excess Primer.

[0156] The process described in the preceding paragraph has severalpotential novel uses: 1) it can be useful for “covering up” a sequencewithin the single stranded molecule that might otherwise “interfere”with a subsequent step, for instance capturing of the moleculesingle-stranded molecule on a solid matrix; 2) it can be used to measureor unfold regions within a single-stranded molecule that exhibitsecondary structures such as hairpins; 3) it can be used to blockProduct Evolution (the phenomenon of Product Evolution is describedbelow); 4) it can be used to enable detection of the single strandedmolecule by staining with at dye, such as SYBR® Green, which can bind tothe double-stranded portion of the single-stranded molecule; 5) it canbe used to increase the rate of single-strand synthesis by extension ofthe Excess Primer; 6) by using a labeled Low-T_(m) primer, this methodcan be used to label the strand complementary to the single-strandedproduct.

[0157] Single-strand to double-strand conversion can be combined withend-point analysis to achieve another form of LATE-PCR end-point assay.In this case the reaction is carried out in the presence of an ExcessPrimer that is fluorescently tagged at its 5′end and an additionaloligonucleotide that is complementary to this primer and blocked with aquenching moiety, such as Dabcyl, at its 3′end. The complementaryoligonucleotide (CO) is designed to have a T_(m[0]) ^(CO) at least 5° C.below the T_(m[0]) ^(X) of the Excess Primer to its target sequence inthe amplicon (see Li et al. 2002) One additional short oligonucleotide,the Low-T_(m) Primer, is also added to the reaction. The Low-T_(m)primer is designed to hybridize to a sequence within the single-strandedproduct of the reaction, when the temperature of the reaction is droppedin the low-temperature step. When the temperature is then slowlyincreased, the DNA polymerase extends the Low-T_(m) Primer, convertingthe 5′end of the single-strand into a double-strand.

[0158] Under these circumstances the fluorescently-tagged Excess Primeris incorporated into every copy of the amplicon strand that it primes,during both the exponential and the linear phase of LATE-PCR. At theend-point of the reaction, when the temperature of the reaction isdropped-and-then-raised, the Low-T_(m) primer is extended and thecomplementary oligonucleotide hybridized to the 5′end of thesingle-strand is displaced. When the reaction temperature is dropped fora final time, the incorporated copies of the Excess Primer fluoresce,while the unincorporated copies of the Excess Primer hybridize to theircomplementary strands that quench their fluorescence, in accord with thefindings of Li et al. (2002). The resulting fluorescence of theincorporated primers can be used as a measure of the number ofsingle-stranded molecules that have been synthesized in the reaction.

[0159] LATE-PCR can also be used to generate single-stranded moleculesin situ, which are then subsequently detected by use of a secondarymethod of amplification, such as rolling circle amplification combinedwith various means of detection the resulting single strands. Thisapplication of LATE-PCR takes advantage of the high level ofprimer-target specificity afforded by PCR, but does not require that thenumber of single-stranded molecules so generated be directly detectable.The secondary method of amplification, which might otherwise generate anunacceptably high rate of false positives, then is used to detect thepresence of the pool of specific single-stranded molecules generated bythe LATE-PCR reaction. As will be recognized by persons skilled in theart, LATE-PCR with secondary amplification is also useful formultiplexing, because it avoids the generation of high concentrations ofLATE-PCR products that might tend to interact.

[0160] When LATE-PCR is combined with a secondary method ofamplification to further amplify, the relationship between ampliconT_(m) ^(A) and Excess Primer, T_(m[0]) ^(X) becomes less constrainingand may exceed 25° C.

[0161] Use of LATE-PCR for Production of Single-Stranded Molecules:

[0162] In addition to assays, LATE-PCR can be used to synthesizesingle-stranded products for any purpose. One such purpose is thegeneration of starting material for subsequent methods of sequencing.Another is production of single-stranded oligonucleotides for use ashybridization probes, for example, in situ hybridization probes.

[0163] Product Evolution During LATE-PCR Amplification:

[0164] Whereas symmetric PCR amplifications tend to plateau and stopafter about 50 cycles, amplifications according to this inventioncontinue to generate single-stranded amplicons for 75 cycles or more. Wehave discovered, however, that in some instances the single-strandedmolecules that accumulate during amplification of some targets tend tointeract and “evolve”, if the thermal cycles are not maintained at ahigh level of stringency. The resulting “derivative molecules” are thenamplified as double-stranded molecules. We call this “ProductEvolution.” Product Evolution is not consistently observed by use of ahybridization probe, such as molecular beacons, because these probestend to hybridize both to the initial single-stranded product of thereaction and to the “derivative molecules” generated by ProductEvolution. However, the process of Product Evolution can be detected andanalyzed by use of SYBR® Green, which stains double-stranded moleculesregardless of their sequence, and by melting point analysis of theresulting products. Product Evolution can be further analyzed byelectrophoresis of the amplified products.

[0165] Product Evolution is stochastic and undesirable, insofar as italters the sequence of the amplified product. This phenomenon has beenreported for asymmetric PCR (Gyllensten and Erlich, (1988)), althoughthe molecular mechanism is unknown at this time. Product Evolution maybe initiated by inappropriate priming of derivative amplicons. FIG. 11illustrates the phenomenon of Product Evolution and demonstrates thatincreasing the stringency of the annealing step in a LATE-PCRamplification or assay delays Product Evolution. Comparison of FIG. 11Aand FIG. 11B shows how the phenomenon of Product Evolution is distinctfrom amplification of non-specific products. Comparisons of the insetsshow how the product can evolve to a higher melt peak undernon-stringent conditions. Curve 111 is the melt analysis of a samplegenerated under stringent conditions and curve 112 shows the kinetics ofamplification under stringent conditions. Curve 113 is the melt analysisof a sample generated under non-stringent conditions, and curve 114shows the kinetic analysis of amplification under non-stringentconditions. Horizontal arrows in the graphs indicate the number ofcycles of single stranded DNA accumulation in the absence of productEvolution; vertical arrows in the insets show the temperature meltingpeak for the correct product.

[0166] It will be recognized by individuals skilled in the art thatthere are a number of ways to increase the stringency of the reactionand thereby suppress inappropriate initiation. The possible ways ofincreasing stringency include:

[0167] a) increasing the annealing temperature to limit thehybridization of the primers, particularly the Excess Primer,

[0168] b) raising or lowering the extension temperature away from theoptimum of the DNA polymerase,

[0169] c) decreasing the concentration of the DNA polymerase,

[0170] d) decreasing the concentration of the Excess Primer,

[0171] e) in assays employing a low temperature detection step, limitingthat step to minimal duration followed by rapid ramping to meltingtemperatures in order to minimize possible primer extension atmismatched sites.

[0172] An alternate explanation to account for Product Evolution is thatit is caused by imperfect annealing of the Excess Primer to a sitewithin either a) the initial genome, or b) the single-strands thataccumulate during the linear phase of LATE-PCR. Imperfect annealingwould not occur if amplification conditions remain stringent but wouldbe favored each time the temperature is dropped during a low temperaturedetection step in an assay. An imperfectly hybridized primer, oncebound, will be extended to the 5, end of a single-strand template. Incase b), the resulting partial strand would then be amplified as a shortdouble-stranded molecule in subsequent thermal cycles, because the sameExcess Primer would prime replication in both directions.

[0173] Product Evolution and non-specific amplification can besuppressed by temporarily decreasing the effective concentration of theExcess Primer and hence, increasing specificity, each time an assayproceeds through the low temperature detection step. This goal can beachieved by inclusion of an oligonucleotide that is complementary to theExcess Primer, but only binds to the Excess Primer when the temperatureof the reaction is reduced below the annealing temperature of the ExcessPrimer to its target sequence in the amplicon. The optimal base sequenceof the complementary oligonucleotide depends on the sequence compositionof the particular Excess Primer and is established experimentally.However, it can be anticipated that the complementary oligonucleotidewill have the following characteristics:

[0174] The concentration-adjusted melting temperature of thecomplementary oligonucleotide, T_(m[0]) ^(CO), should be at least 3° C.below T_(m[0]) ^(X). This can either be achieved by decreasing theconcentration of the complementary oligonucleotide relative to theExcess Primer, or by altering the length or the sequence of thecomplementary oligonucleotide relative to the Excess Primer. However, itis most desirable to keep the concentration of the complementaryoligonucleotide in excess of the concentration of the Excess Primer, sothat when the temperature of the reaction is dropped below T_(m[0])^(CO), the majority of Excess Primer molecules will be hybridized tocomplementary oligonucleotide molecules. It is therefore most desirableto either shorten the complementary oligonucleotide relative to theExcess Primer, or to deliberately mismatch the complementaryoligonucleotide of the Excess Primer or both. Most preferably, thecomplementary oligonucleotide can be shortened at its 3′ end. At leastthe three bases at the 5′ end of the complementary oligonucleotideshould be perfectly matched to the three bases at the 3′ end of theExcess Primer to prevent the Excess Primer from initiating strandreplication. Further, the 3′ end of the complementary oligonucleotideshould be blocked by a modification such as a phosphate group to ensurethat the complementary oligonucleotide cannot act as a primer.

[0175] Assays that utilize amplification according to this inventioninclude homogenous end-point assays and homogenous real-time assays. Inhomogenous assays, no separation of products is required. Generallytubes of the amplification reaction need not be opened, as detectionmeans, for example fluorescent dyes or fluorescent probes, can be addedprior to the start of amplification. Assays according to this inventionmay utilize either 2-step PCR or 3-step PCR. Certain preferredembodiments additionally include a low temperature detection stepfollowing primer extension. If a low temperature detection step is used,it need not be included in the early cycles of amplification andpreferably is omitted until 5-10 cycles prior to the C_(T) to promotespecificity and suppress Product Evolution, discussed above, while stillmaking it possible to establish background levels of fluorescence priorto C_(T).

[0176] LATE-PCR Assays Combined with Harvesting of Single-StrandedProducts

[0177] Whether LATE-PCR is carried out for the purpose of generating asingle amplicon or many amplicons, it is desirable to minimizeinappropriate interaction of single-stranded molecules in order toreduce the chance that Product Evolution will occur as the concentrationof these molecules increases. Periodic capture and removal ofsingle-stranded molecules from an ongoing reaction, during selected orall linear amplification cycles, provides a simple and versatile meansof keeping the concentration of the products low. Capture and removal ofsingle-stranded molecules can be accomplished in a variety of devicesand formats. For example, a LATE-PCR amplification can be carried out ina “racetrack” like chamber around which the reactants repeatedly cycle.The speed at which the reactants rotate around the racetrack can becontrolled, and adjacent sections of the racetrack can be differentiallyheated or cooled to achieve the required pattern of thermal cycling. Onesector of the racetrack, or several adjacent sectors of the racetrack,can have surfaces that include covalently linked capture probes with oneor more sequences complementary to one or more single-stranded products.As the reactants pass through these sectors the temperature of thereaction can be cooled, as in a low-temperature detection step. Underthese conditions, each single-stranded molecule will hybridize to itsparticular capture probes, while double-stranded template molecules, aswell as Taq DNA polymerase (and other proteins) and all of the smallmolecules of the reaction mixture move onward to the next chamber in theracetrack. Once the reactants have cleared the capture-probe sector ofthe racetrack, it can be isolated and its temperature raised to releaseand recover the single-stranded molecules for subsequent analysis ormanipulation. Persons skilled in the art will appreciate that the aboveprinciples can be applied to additional systems and devices designed toharvest the single-stranded products of a LATE-PCR.

[0178] It is anticipated that capture and removal of the single-strandedproducts of a LATE-PCR reaction will permit repeated rounds of productsynthesis well beyond the number of rounds observed in a typicalsymmetric PCR reaction. For instance, we have shown that some LATE-PCRassays can be sustained for at least 100 thermal cycles in a closed tubereaction of 25-100 μl. This means that neither the Taq DNA polymerase,nor the reporter dye, nor the Excess Primer, nor the nucleotideprecursors necessarily becomes limiting in a LATE-PCR reaction. Thisobservation stands in contrast to the commonly held view in thescientific literature for symmetric PCR. For instance Liu and Saintwrite, “Since the reaction is performed in a closed tube containing asmall amount of reaction mixture (25-50 μl), the reaction kinetics canbe affected by all components in the reaction mixture, includingreporter dye, nucleotide concentration, primer concentration, andinitial copy number (or concentration) of template.

[0179] Because the reporter dye, nucleotide, primer concentration, andenzymatic activity can become limiting to the rate of synthesis ofamplicon, the rate of synthesis of amplicon will slow and eventuallycease.” (Liu, W. & Saint, D. A. (2002) “A New Quantitative Method ofReal Time Reverse Transcription Polymerase Chain Reaction Assay Based onSimulation of Polymerase Chain Reaction Kinetics”, AnalyticalBiochemistry 302: 52-59).

[0180] In contrast, our results demonstrate that the limits onamplification according to this invention are due to the fact that theconcentration of the amplicon strand(s) reaches levels that allow themto effectively compete with their own primers (particularly theextension product of the Excess Primer competes with the Excess-Primer)for hybridization to the same target molecules. As shown earlier in theapplication, one means of sustaining production of the single-strandproduct is to optimize (T_(m) ^(A)−T_(m[0]) ^(X)) to the range of 7-25°C., most preferably 12° C. However, optimization of (T_(m) ^(A)−T_(m[0])^(X)) is not always possible, for instance if the amplicons are GC-richor very long. Under these circumstances, capture and removal of theaccumulating single strands can serve as a substitute for maintainingthe specified melting-point differential between amplicon and ExcessPrimer. This enables the LATE-PCR to go on synthesizing single-strandsfor many cycles.

[0181] In another embodiment of the invention the LATE-PCR can becarried out under conditions in which one of the primers, mostpreferably the Excess Primer, is fixed to a solid matrix or surface suchthat each cycle of primer extension results in construction of anextended primer strand which remains attached to the solid surface, forexample, a bead or the wall of the reaction chamber. It is anticipatedthat under these conditions T_(m[0]) of the attached primer will beadditionally dependent on the fact that the primer is not freelydiffusible, as well as by the packing density of the primer on thesurface, by the volume, and by the geometry of the space in which thereaction takes place. Therefore, the T_(m[0]) of the primer, forinstance T_(m[0]) ^(X), will have to be determined empirically under theexperimental conditions of the reaction.

EXAMPLES Example 1

[0182] Design of Primer Pairs, Tay Sachs HEX-A Gene

[0183] Rather than starting with an existing matched pair of symmetricPCR primers and making modifications to achieve a primer pair accordingto this invention, we prefer to design primer pairs using availablecomputer software. We have successfully utilized the computer programOligo® 6.0 (Oligo® Primer Analysis Software Manual, version 6.0 forWindows, Molecular Biology Insights, Inc. Sixth Edition, March 2000) toidentify candidate primer pairs. To determine T_(m[0]) for candidateprimers using the “Nearest-Neighbor” method, we have successfullyutilized the formula provided in the previous sections that relies onthe Allawi and SantaLucia (1997) values for enthalpy and entropy. Wedesigned a LATE-PCR primer pair for the identification of the wild-typeand 1278+TATC allelic sequences in exon 11 of the alpha subunit of thebeta-N-acetylhexosaminidase (HEX-A) gene. These alleles are associatedwith Tay-Sachs disease. The mutant allele, 1278+TATC, accounts for82-90% of Tay-Sachs carriers within the Ashkenazi Jewish population.(For a recent review see Advances in Genetics, vol. 44, edited byDesnick and Kaback (2001), which is entirely devoted to Tay-Sachsdisease). The Oligo® 6.0 program was used to identify a set ofcompatible primers for symmetric PCR amplification of a segment in HEX-Aexon 11 containing nucleotide position 1278 (GenBank accession #: NM000520). The search parameters were set to identify pairs of primersthat would generate amplicons smaller than 100 base pairs. Among thecandidate primer pairs, we chose a primer set whose sequences are givenbelow in Table IV. According to the default settings of Oligo® 6.0 theseprimers have matching T_(m)'s (upper primer: 73.6° C.; lower primer:73.0° C.). We then proceeded to calculate the primer T_(m[1]) values ata standard concentration of 1 μM and to calculate the T_(m[0]) valuesfor the Excess and the Limiting Primers at concentrations of 1 μM and 25μM, respectively (1:40 primer ratio; the monovalent cation concentrationfor these calculations was set to 0.07 M) using the Nearest Neighborformula, as stated earlier. For convenience, the calculations ofenthalpy and entropy with Allawi and SantaLucia (1997) nearest neighborvalues can be done using the computer program, MELTING (Le Novère, N.(2001) “MELTING, Computing the Melting Temperature of Nucleic AcidDuplex,” Bioinformatics 17: 1226-7). The results are given in Table IV.The above primer ratio and concentrations were chosen based on trialsinvolving monitoring each amplicon strand with molecular beacons duringasymmetric amplification and revealed that at 25 nM the Limiting Primerbecomes depleted shortly after the reaction reaches the threshold cycle(C_(T)). The C_(T) is thus reached before exponential amplificationstops and linear amplification begins.

[0184] The Excess Primer was used at 1 μM to promote maximal synthesisof single-stranded DNA during the linear-phase of LATE-PCR. Table Vshows the calculated T_(m[1]) and T_(m[0]) values. The matching T_(m[1])values makes this primer set suitable for symmetric PCR. The fact thatT_(m[0]) ^(L)<T_(m[0]) ^(X) makes this primer set unsuitable for use inLATE-PCR amplifications and assays. TABLE IV Initial primer pairsuggested by Oligo ® 6.0 SEQ ID Primer Sequence NO: Conc. T_(m[1]) Conc.T_(m[0]) TSD1242S20 5′-CCTTCTCTCTGCCCCCTGGT-3′ 1 1 μM 64.8° C. 25 nM58.9° C. TSD1301A22 5′-GCCAGGGGTTCCACTACGTAGA-3′ 2 1 μM 64.3° C.  1 μM64.3° C.

[0185] The selected primers were then altered to meet criteria requiredfor LATE-PCR amplification. Primer TSD1242S20 was lengthened at its 5′end using the endogenous HEX-A sequence, and Table V shows the results.In Table V, the nucleotides in the primer sequences that are presentedin bold correspond to the sequences in Table IV. The two nucleotides inregular font at the 5′ end of the Limiting Primer are the addednucleotides.

[0186] The original pair of primers listed in Table IV generate an 81bp-long amplicon whose T_(m) ^(A) is 78.6° C. (according to % GC method:T_(m) ^(A)=81.5+16.6 log [M]/(1+0.7[M])+0.41 (% G+% C)−500/length, at a70 mM salt concentration: (Wetmur, J. G. (1991). “Applications of thePrinciples of Nucleic Acid Hybridization, Crit Rev Biochem Mol Biol 26:227-259.) The modified primers used for LATE-PCR, Table V, generate anamplicon 83 bp-long that has a T_(m) ^(A)=79.2° C. The difference (T_(m)^(A)−Tm[0]^(X)) is 15° C. (79.2−64.3=14.9, which rounds to 15° C.). Thisprimer pair thus satisfies the conditions (T_(m) ^(L)_([0])−T_(m[0]) >)0 and (T_(m) ^(A)−T_(m) ^(X) _([0]))<18° C. TABLE VPrimers modified to meet LATE-PCR specifications SEQ ID Primer SequenceNO: Conc. T_(m[1]) Conc. T_(m[0]) TSD1240S225′-GCCCTTCTCTCTGCCCCCTGGT-3′ 3 1 μM 69.4° C. 25 nM 64.0° C. TSD1301A225′-GCCAGGGGTTCCACTACGTAGA-3′ 2 1 μM 64.3° C.  1 μM 64.3° C.

Example 2

[0187] Design of Primer Pairs, Human Beta Globin Gene

[0188] Another primer design is provided by the choice of primers forthe detection of specific mutations in the human beta globin. Thelocation of the primer sequences was chosen such that sites of theIVSI-110 and the codon 39 mutations known to cause beta thalassemia wereincluded in the amplicon. The possible location of the 3′ end of eachprimer was limited to regions without homology to the other members ofthe globin gene family to insure that the beta globin gene would bepreferentially amplified. Once prospective sites were identified, thosesequences were examined using the Oligo® 6.0 software and the regionlikely to yield a primer with a higher T_(m[1]) was chosen for theLimiting Primer, in this case the lower strand sequence. A concentrationof 50 nM was chosen for the Limiting Primer and the T_(m[0]) ^(L) ofpossible primers of different lengths was determined as described inExample 1. The T_(m[0]) ^(X) of possible Excess Primers at aconcentration of 1000 nM was determined in the same manner. A LimitingPrimer 26 nucleotides long with a T_(m[0]) ^(L)=66° C. was initiallyselected, and an Excess Primer 28 nucleotides long with a T_(m[0])^(X)=66° C. was chosen, that T_(m[0]) ^(X) being 15 degrees below theT_(m) ^(A) of 81° C. (amplicon of 191 base pairs, 52.4% GC). Byincluding one A-to-G modification near the 5′ end of the initiallyselected Limiting Primer and by increasing its length to 30 nucleotideswe obtained a final Limiting Primer having a T_(m[0]) ^(L)=72° C.

Example 3

[0189] Design of LATE-PCR Primers for Cystic Fibrosis Gene

[0190] The criteria described herein for (T_(m[0]) ^(L)−T_(m[0]) ^(X))and for (T_(m) ^(A)−T_(m[0]) ^(X)), alone and in combination, havedemonstrable effects on PCR amplification. We have demonstrated certaineffects utilizing primers, which we designate “CFTR” primers foramplifying the genomic sequence surrounding the Δ508 mutation, the mostcommon cause of cystic fibrosis. For the tests reported in this examplewe have utilized Limiting Primers and Excess Primers from among thoselisted below in Table VI, which sets forth for each primer itsnucleotide sequence and its T_(m[0]) for a Limiting Primer concentrationof 50 nM and an Excess Primer concentration of 1000 nM. In testsutilizing a hybridization probe against the single-stranded amplicongenerated by extension of the Excess Primer, we used a molecular beaconmodified with a quencher on one end and a fluorophore on the other end,having the following sequence 5′ FAM-CGCGCTTATCATCTTTGGTGTTTCCTATAGCGCG—Dabcyl 3′ (SEQ ID NO: 9) where thesix nucleotides at each end (underlined) form the stem and thereforewere not used to calculate T_(m[0]) ^(P). This probe had a T_(m[0]) ^(P)of 56° C. empirically measured under conditions that included 3 mMmagnesium, 600 nM molecular beacon and 600 nM target. TABLE VI CFTRLimiting and Excess Primer Sequences SEQ ID Primer Name Sequence NO:T_(m[0]) Limiting Primers: CF403 S18 GATTATGCCTGGCACCAT  4 51.1 CF402S19t TGATTATGCCTGGCACCAT 10 53.4 CF402 S19 GGATTATGCCTGGCACCAT  6 54.1CF400 S21t CTTGATTATGCCTGGCACCAT 11 55.2 CF401 S20 TGGATTATGCCTGGCACCAT12 56.2 CF400 S21 CTGGATTATGCCTGGCACCAT 13 57.1 CF399 S22CCTGGATTATGCCTGGCACCAT  7 59.5 CF398 S23 TCCTGGATTATGCCTGGCACCAT 14 60.9CF392 S29 CAGTTTTCCTGGATTATGCCTGGCACCAT 15 64.1 CF391 S30TCAGTTTTCCTGGATTATGCCTGGCACCAT 16 65.0 Excess Primers: CF475 A16GACGCTTCTGTATCTA 17 47.2 CF476 A17 TGACGCTTCTGTATCTA 18 49.9 CF477 A18ATGACGCTTCTGTATCTA 19 50.7 CF479 A20 TGATGACGCTTCTGTATCTA 20 54.2 CF482A23 CTTTGATGACGCTTCTGTATCTA  5 56.4 CF483 A24 GCTTTGATGACGCTTCTGTATCTA21 59.0 CF488 A29 GGCATGCTTTGATGACGCTTCTGTATCTA 22 65.0

[0191] A first demonstration is reported in Table VII and FIGS. 2A and2B. Two series of five PCR amplifications each were performed utilizingExcess Primer CF 479 A20 and one of five Limiting Primers identified inTable VII. In the first series all amplifications utilized the sameannealing temperature, 52° C. (2° C. below T_(m[0]) ^(X)). In the secondseries all amplifications used an annealing temperature 2° C. belowT_(m[0]) ^(L). All amplifications were monitored with SYBR® Green, afluorescent dye that binds to double-stranded DNA and, thus, monitorsthe production of double-stranded amplicon during the initial phase ofamplification when both primers are present.

[0192] Table VII sets forth the difference (T_(m[0]) ^(L)−T_(m[0]) ^(X))rounded to the nearest whole number following subtraction. Table VIIalso sets forth the mean C_(T) from three replicates when the annealingtemperature was 52° C. (first series) and when the annealing temperaturewas 2° C. below T_(m[0]) ^(L) (second series). The fluorescence readings(average of three replicates) from the first series are set forth inFIG. 2A (first series) and FIG. 2B (second series). TABLE VII Effect of(T_(m[0]) ^(L) − T_(m[0]) ^(X)) Annealing Annealing TemperatureTemperature Limiting T_(m[0]) ^(L) − T_(m[0]) ^(X) First Series MeanSecond Mean Primer ° C. ° C. C_(T) Series ° C. C_(T) CF403 S18 −3 5235.1 49 34.4 CF402 S19 0 52 34.7 52 34.2 CF400 S21 +3 52 33.8 54 34.7CF399 S22 +5 52 32.0 57 32.4 CF398 S23 +7 52 30.8 59 32.4

[0193] Amplification mixtures included 1000 nM Excess Primer, 50 nMLimiting Primer, 0.4 mM each dNTP, 0.2×SYBR® Green (Molecular Probes),3.5 mM MgCl₂, 0.06 Units/μl Platinum Taq DNA Polymerase (Invitrogen),1×PCR buffer (20 mM Tris-HCl (pH 8.4), 50 mM KCl) and 1× AdditiveReagent (1 mg/ml BSA, 750 mM trehalose, 1% Tween-20) and 600 picogramsof human genomic DNA in a total volume of 25 μl. Amplification andfluorescence detection were done using a Cepheid Smart Cycler thermalcycling instrument with real-time fluorescence detection. An initialdenaturation step of 3 minutes at 95° C. was followed by 60 cycles of:95° C. for 5 seconds, 52° C. (or other specified annealing temperature)for 15 seconds, and 72° C. for 15 seconds with fluorescence acquisition.

[0194]FIG. 2A shows the mean real time fluorescence increase in samplesfrom the first series. Each curve corresponds to reactions using primerswith different (T_(m[0]) ^(L)−T_(m[0]) ^(X)) values as listed in TableVII. The earliest detection (lowest mean C_(T) value) was obtained usingthe primer pair with the highest value (T_(m[0]) ^(L)−T_(m[0]) ^(X))(+7, see curve 21). Mean C_(T) values increased with each decrease inthe value of (T_(m[0]) ^(L)−T_(m[0]) ^(X)) (+5, curve 22; +3, curve 23;+0, curve 24; −5, curve 25). Lower C_(T) values demonstrate a higherrate of amplification (i.e., increased efficiency) during theexponential phase of the reaction. All samples eventually reachedsimilar final fluorescence, that point corresponding to the completionof double-stranded DNA synthesis. (The continued synthesis ofsingle-stranded DNA is not detected using this method.)

[0195] Gel electrophoresis revealed that each sample for which the valueof (T_(m[0]) ^(L)−T_(m[0]) ^(X)) was between 0 and +7 yielded a similarquantity of specific amplicon. However, one of the three samples with(T_(m[0]) ^(L)−T_(m[0]) ^(X)) equal to −3 had considerably less specificamplicon and contained large amounts of non-specific products. Theaverage level of non-specific product was lower in samples with(T_(m[0]) ^(L)−T_(m[0]) ^(X)) equal to 0 or +3, and extremely low insamples with (T_(m[0]) ^(L)−T_(m[0]) ^(X)) equal to +5 or +7. Theresults demonstrate that the increase in T_(m[0]) ^(L) several degreesabove the annealing temperature determined as optimal for the ExcessPrimer does not increase the amount of detectable mis-priming by theLimiting Primer, and in fact, increases the specificity of the reaction.

[0196]FIG. 2B shows the results of samples with the same primer pairs asabove, but with annealing done at 2° C. below T_(m[0]) ^(L). Again, eachcurve corresponds to reactions using primers with different (T_(m[0)^(L)−T_(m[0]) ^(X)) values as listed in Table VII. Curves 26 and 27,corresponding to samples where (T_(m[0]) ^(L)−T_(m[0]) ^(X)) equal to +5or +7, respectively have a mean C_(T) value that was lower than sampleswith delta-T_(m) equal to +3 or lower (+3, curve 28; +0, curve 29; −3,curve 210) indicating the higher amplification efficiency in the former.Comparison of the mean C_(T) values obtained at the different annealingtemperatures for each primer pair shows that increasing the annealingtemperature above the Excess Primer T_(m[0]) results in only slightincrease in mean C_(T) values. However, the increased annealingtemperature improves the specificity of the reaction even further,reducing the average level of non-specific product that is observedusing gel electrophoresis. Conversely, lowering the annealingtemperature of the primer pair with (T_(m[0]) ^(L)−T_(m[0]) ^(X)) equalto −3 results in a slight reduction in mean C_(T) value, but the averageamount of specific amplicon was reduced and the average amount ofnon-specific product was increased. The reduced specificity of thereaction is presumably due to mis-priming by the Excess Primer at thelower annealing temperature. Thus, one advantage of having T_(m[0])^(L)>T_(m[0]) ^(X) is that the true optimal annealing temperature forboth primers can be set, sufficiently low to allow high amplificationefficiency from the Limiting Primer, but sufficiently high to limitmis-priming from the Excess Primer that can generate non-specificproducts.

[0197] PCR reaction mixtures containing one of three of the LimitingPrimers in Table VII, namely CF 403 S18, CF 402 S19 and CF 399 S22, butincluding a molecular beacon probe rather than SYBR® Green, weresubjected to both series of amplifications. The first series (annealingtemperature 52° C.) yielded mean C_(T) values of 38.9, 37.6 and 36.8,respectively. The second series (annealing temperature 2° C. belowT_(m[0]) ^(L)) yielded mean C_(T) values of 38.5, 38.6 and 38.9,respectively. When annealing was done at 2 degrees below T_(m[0]) ^(L),the lowest mean C_(T) value was obtained for samples with (T_(m[0])^(L)−T_(m[0]) ^(X))=+5 and the highest mean C_(T) value was obtained forsamples with (T_(m[0]) ^(L)−T_(m[0]) ^(X))=−3, verifying the fact thatamplification efficiency increases when Limiting Primer T_(m[0]) ^(L) israised. Subsequent fluorescence increase was at similar rates in allgroups.

Example 4

[0198] Design of Efficient LATE-PCR Primers

[0199] A set of PCR primers and a molecular beacon probe were designedfor the ΔF508 allele of the cystic fibrosis gene based on published genesequences. (Riordan et al. (1989) “Identification of the Cystic FibrosisGene: Cloning and Characterization of the Complementary DNA,” Science245: 1006-73). The primer and molecular beacon sequences were: upperprimer: 5′-CCTGGATTATGCCTGGCACCAT-3′ (SEQ ID NO: 7) lower primer:5′-CCTGATGACGCTTCTGTATCTA-3′ (SEQ ID NO: 8) molecular beacon probe:5′-TET-CGCGCTAAAATATCATTGGTGTTTCCT (SEQ ID NO: 23) AAGCGCG-DABCYL-3′

[0200] where the underlined terminal sequences in the probe form ahairpin stem.

[0201] Primers were analyzed and varied to have difference T_(m)'sutilizing Oligo® 6.0 software. In this way we obtained primer pairs thatthe program (as is usual, in default mode) had calculated T_(m)'s asfollows: either no difference in T_(m) (both 65° C.; FIG. 3A), a 5° C.difference in T_(m) (upper primer 70° C., lower primer 65° C.; FIG. 3B),or a 10° C. difference in T_(m) (upper primer 75° C., lower primer 65°C.; FIG. 3C). Additionally, the primers were either equimolar (both 500nM, curves 32, 34, and 36) or present at a 1:10 ratio (50 nM upperprimer:500 nM lower primer, curves 31, 33, and 35). Fifteen microlitersof concentrated PCR reagent mixture were added to each tube containing alysed cell to yield a final sample volume of 25 microliters with finalconcentrations of 1×PCR buffer (Invitrogen, Carlsbad, Calif., USA), 3.75mM MgCl₂, 0.25 mM dATP, 0.25 mM dCTP, 0.25 mM dGTP, 0.75 mM dUTP,primers as indicated, 1.2 μM molecular beacon, and 1.5 units PlatinumTaq DNA polymerase (Invitrogen). Amplification and fluorescencedetection were carried out in an ABI 7700 thermal cycling instrumentwith real-time fluorescence detection (Applied Biosystems, Foster City,Calif., USA). Thermal cycling consisted of an initial 5-minutedenaturation at 95° C. followed by 4 cycles of 95° C. for 10 seconds,55° C. for 2 minutes, and 72° C. for 30 seconds, followed by 21 cyclesof 95° C. for seconds, 55° C. for 30 seconds, and 72° C. for 30 seconds,followed by 35 cycles of 95° C. for 10 seconds, 52° C. for 30 seconds,and 72° C. for 30 seconds with fluorescence acquisition during the 52°C. step. Molecular beacons specific for the ΔF508 allele and for thenormal allele were included in each reaction and were targeted to thelower primer-strand. Amplification and fluorescence detection werecarried out in an ABI Prism 7700 Sequence Detector-Results are shown inFIGS. 3A-3C. Results are plotted as the cycle number (X-axis) vs. themolecular beacon delta fluorescence units (Y-axis). FIG. 3A shows theresults of replicates amplifications with the equal T_(m) primers (65°C., 65° C.) in a symmetric PCR amplification having 1:1 ratio of primers(curve 32) and in an asymmetric amplification having a 1:10 ratio (curve31). FIG. 3B shows the results of replicate amplifications with primershaving T_(m)'s differing by 5° C. (70° C., 65° C.) with a 1:1 ratio ofprimers (curve 44) and with a 1:10 ratio (curve 33). FIG. 3C shows theresults of replicate amplifications with primers having T_(m)'sdiffering by 10° C. (75° C., 65° C.) with a 1:1 ratio of primers (curve36) and with a 1:10 ratio (curve 35).

[0202] The asymmetric reaction (1:10 primer ratio, curve 31) using equalT_(m) primers results in a fluorescence signal that is delayed (laterC_(T)), as compared to the symmetric reaction (equimolar primers, curve32) (FIG. 3A). However, when a 5° C. difference in T_(m) is introduced(FIG. 3B), the C_(T) for the primers with a 1:10 ratio (curve 33) occursmuch earlier, almost as early as for the equimolar primers (curve 34).Additionally, the final fluorescence signal for the primers with a 1:10ratio (curve 33) is much higher than the signal for the equimolarprimers (curve 34), and it has not plateaued, even at 60 cycles. When a10° C. difference in T_(m) (FIG. 3C) is introduced, the C_(T) for theprimers with a 1:10 ratio (curve 35) is the same as for the equimolarprimers (curve 36), and the final fluorescence is much higher and doesnot plateau.

Example 5

[0203] Designing Primers Based on the Relationship between T_(m[0]) ^(X)and T_(m) ^(A)

[0204] LATE-PCR also takes into account the difference between T_(m[0])^(X) and T_(m) ^(A). T_(m) ^(A) is higher than T_(m[0]) ^(X), but if thedifference between these two values is too great, then lower amounts ofsingle-stranded product will be generated. A demonstration of this isreported in Table VIII and FIG. 6. We have demonstrated this using“CFTR” primer pairs for which (T_(m[0]hu L)−T_(m[0]) ^(X))=0° C., butthose values are different in each set of replicate samples, and varywith respect to T_(m) ^(A). PCR amplification mixtures were prepared asdescribed in Example 3, with molecular beacon probe added at aconcentration of 600 nM rather than SYBR® Green. The thermal cyclingprofile was also the same, except that annealing was at 2° C. belowT_(m[)0]^(L) for the first 25 cycles, then shifted to 52° C. for anadditional 50 cycles in order to monitor molecular beacon fluorescenceunder equivalent conditions for all samples. TABLE VIII/ Effect ofvarying (T_(m) ^(A) − T_(m[0]) ^(X)) for (T_(m[0]) ^(L) − T_(m[0]) ^(X))= 0 (T_(m) ^(A) − T_(m) ^(A) T_(m[0]) ^(X)) early slope late slopeLimiting Power Excess Primer ° C. ° C. Mean C_(T) (cycles 45-60) (cycles60-75) CF403 S18 CF477 A18 70 19 38.0 5.8 4.3 CF402 S19 CF479 A20 70 1638.9 8.0 5.6 CF401 S20 CF482 A23 70 14 37.4 9.1 6.6 CF399 S22 CF483 A2471 12 37.4 10.2 5.9 CF391 S30 CF488 A29 72 7 36.8 8.0 6.6

[0205] The average molecular beacon fluorescence for each group of 3replicate samples is shown in FIG. 6. Each curve in FIG. 6 correspondsto a value (T_(m) ^(A)−T_(m[0]) ^(X)) from Table VIII. Mean C_(T) valuesand rates of signal increase (slopes) are presented in Table VIII.Samples with (T_(m) ^(A)−T_(m[0]) ^(X))=12° C. (curve 61) yielded thestrongest beacon signal and presumably the largest quantity ofsingle-stranded CFTR product in this series. Samples with (T_(m)^(A)−T_(m[0]) ^(X))=19° C. (curve 65) yielded the lowest signal. Sampleswith intermediate values of (T_(m) ^(A)−T_(m[0]) ^(X))=14° C. (curve62), or 16 (curve 64) yielded intermediate average signal intensitycorresponding with that value. Samples with (T_(m) ^(A)−T_(m[0])^(X))=7° C. (curve 63) also yielded intermediate final signal intensity,but displayed different kinetics than the other groups; the fluorescenceremained relatively low for several cycles following initial detection,but the average rate of increase (slope) was among the highest duringthe final 15 cycles, suggesting that the Excess Primer in those samplescontinued to amplify efficiently as the concentration of the competingproduct strand increased. Such results may be advantageous forapplications that require continued synthesis of the single strandedamplicon without generating non-specific product. Note that although themajority of samples (all groups) showed continued fluorescence increaseto cycle 75 with only slightly reduced slopes, a few individual samplesdisplayed greatly reduced slope or reached a plateau during the last 5to 10 cycles. This may be due to Product Evolution, or to the generationof non-specific product in samples with matched primer T_(m[0]).

[0206] The benefits of simultaneously optimizing (T_(m[0]) ^(L)−T_(m[0])^(X)) and (T_(m) ^(A)−T_(m[0]) ^(X)) are illustrated in Table IX andFIG. 7. Each pair of primers in this experiment was designed such that(T_(m[0]) ^(L)−T_(m[0]) ^(X))=+5° C. to +6° C. The values for (T_(m)^(A)−T_(m[0]) ^(X)) ranged from +13 to +23. Curves in FIG. 7 correspondto amplification curves using primers with the values of (T_(m)^(A)−T_(m[0]) ^(X)) listed in Table X. Sample preparation,amplification, and detection were done as described above. TABLE IXEffects of Varying (T_(m) ^(A) − T_(m[0]) ^(X)) for (X_(m[0]) ^(L) −T_(m[0]) ^(X)) = +5-6 early slope late slope Limiting Primer ExcessPrimer T_(m) ^(A) T_(m) ^(A) − T_(m[0]) ^(X) Mean C_(T) (cycles 45-60)(cycles 60-75) CF402 S19t CF475 A16 70 23 43.0 2.3 3.6 CF400 S21t CF476A17 70 20 41.3 4.3 4.3 CF401 S20 CF477 A18 70 19 38.7 4.2 3.8 CF399 S22CF479 A20 71 17 38.5 7.6 5.7 CF392 S29 CF483 A24 72 13 38.1 11.3 7.6

[0207] It is evident from the kinetic plots in FIG. 7 and Table IX thatthe highest molecular beacon signals (cycles 35-60) were in samples with(T_(m) ^(A)−T_(m[0]) ^(X))=13 (curve 71), indicating efficient singlestrand synthesis. The mean intensity of the molecular beacon signaldecreased with each increase in (T_(m) ^(A)−T_(m[0]) ^(X)) to values of17 (curve 72), 19 (curve 73), 20 (curve 74), and 23 (curve 75). Incontrast to the series in the FIG. 6, none of these samples showed anamplification plateau, illustrating another advantage of having(T_(m[0]) ^(L)−T_(m[0]) ^(X))≧+5° C. Electrophoresis of these samplesrevealed only the specific single- and double-stranded amplicon.Non-specific product was not detected, even in the sample group forwhich annealing temperature was lowered from 59° C. to 52° C. for cycles26 to 75.

Example 6

[0208] Kit for Real-Time LATE-PCR Assay

[0209] A LATE-PCR reagent has been designed for use in the detection ofthe normal and ΔF508 alleles of the human cystic fibrosis gene duringpreimplantation genetic diagnosis (PGD). The kit is modular; that is, itcontains DNA polymerase in one package and all other reagents andmaterials in another package. It will be appreciated that the primersand probes together comprise an oligonucleotide set, which can bemarketed as a separate product. The kit, its use, and the assayperformed with the kit, which we call the “CFΔ508 Kit,” are described inthis example in a format that might appear on a product insertaccompanying the kit.

[0210] A Diagnostic Test to genotype a Diploid Human Cell at the F508region of the CFTR gene

[0211] 10 Assay Kit

[0212] For In Vitro Diagnostic Use Only

[0213] IMPORTANT: Read all instructions before starting this test.

[0214] Intended Use

[0215] The CFΔF508 Kit is designed to demonstrate whether one or aplurality of nucleated diploid human cells are genetically homozygousnormal (Normal/Normal), heterozygous (Normal/ΔF508), or homozygousaffected (ΔF508/ΔF508). These determinations are carried out in vitro bycollecting and testing one or more cells, or the DNA derived from suchcells. The knowledge derived from such tests can be used to makedecision about the life of an individual or the healthcare management ofsuch an individual. For instance-those carried out on a single cell froma human embryo, or the cells of a fetus may help the prospective parentsdecide whether or not a particular embryo should be implanted, orwhether or not termination of a pregnancy should be considered. Inanother instance, postnatal knowledge about an individual's genotype canbe used to help optimize the healthcare and life style of saidindividual.

[0216] Explanation

[0217] Cystic fibrosis (CF) is the most common inherited disease amongCaucasian populations with an incidence of about 1 in 2500 births (Welshet al., 1995). The conditions caused by mutations in the CF gene, whichfunctions as a chloride channel in the lungs and other tissue. Mutationsin the CF gene have phenotypes that range from mild to life threateningA 3-basepair deletion within the CF gene, designated ΔF508, accounts fornearly 70% of CF cases and causes severe manifestations of the disease.It results in the absence of phenyalanine at position 508 of the cysticfibrosis transmembrane conductance regulator protein (CFTR) and thiserror prevents normal processing and translocation of the polypeptidechain to apical membranes of epithelial cells (Cheng et al., 1990). Thefirst tests for ΔF508 in single cells used nested PCR to amplify therequisite sequence followed by verification of the final product byeither restriction enzyme digestion (Coutelle et al., 1989),hybridization to allele-specific oligonucleotides (Wu et al., 1993), orheteroduplex formation (Liu et al., 1993; Avner et al., 1994). The firstclinical reports of PGD for CF also utilized heteroduplex analysis ofthe PCR products (Handyside et al., 1992; Verlinsky et al., 1992; Ao etal., 1996). More recent PCR assays have used fluorescently labeledprimers to increase sensitivity and reduce the rate of allele drop out(ADO), a failure to amplify one allele from a heterozygous cell (Findlayet al., 1995; Verlinsky and Kuliev, 2000; Goossens et al., 2000). Whenthis approach is employed, the fluorescently labeled products areseparated and identified by capillary electrophoresis after PCRamplification is finished.

[0218] Couples in which both individuals carry a mutant copy of theΔF508 allele have a one in four chance of having an afflicted child. Onediagnostic alternative available to such couples, known aspreimplantation genetic diagnosis (PGD), offers such couples anopportunity to determine the genetic composition of their embryos beforestarting a pregnancy. If one or more embryos tests negative for theΔF508 allele or heterozygous for the ΔF508 allele the couple than has anopportunity to start a pregnancy based on knowledge that they have avery low probability of having an afflicted fetus or baby. However, PGDis technically difficult because each assay has to be carried out on asingle cell recovered from a cleavage-stage embryo. Prenatal diagnosisprovides an alternative to PGD. Prenatal diagnosis for CF is carried outon amniotic cells recovered by amniocentesis, a technique for collectingamniotic fluid and cells surrounding a fetus in an ongoing pregnancy. Afetus afflicted with CF can be aborted, if the women so chooses withinthe second trimester of her pregnancy. Alternatively, CF can be testedfor and diagnosed postnatally using blood cells and/or other types ofcells. Diagnosis of an afflicted individual is very important forproviding rapid and optimal healthcare.

[0219] In this kit, we describe the use of a LATE-PCR, real-time assaywith molecular beacons to identify the normal and ΔF508 alleles ofcystic fibrosis in single human cells.

[0220] Principle of the Method

[0221] The kit assay makes use of a fluorescently-labeled DNA probeknown as a Molecular Beacon to detect specific DNA sequences amplifiedvia a modified form of the polymerase chain reaction (PCR)^(1,2),hereafter known as LATE-PCR. The kit contains two specific MolecularBeacon Probes, one that fluoresces yellow and is configured to hybridizeto the normal allele of the CFTR gene and one that fluoresces red and isconfigured to hybridize to the ΔF508 allele of the CFTR gene. EachMolecular Beacon Probe has a 6 base-pairs long stem and only fluorescesin the presence of its specific target sequence. A single nucleotidemismatch is sufficient to prevent fluorescence of the Molecular BeaconProbe. LATE-PCR reactions begin with symmetric amplification of bothstrands and then abruptly switch to linear amplification of a singlestrand. Because all copies of the accumulating target strand aresingle-stranded, they are readily detected with a Molecular BeaconProbe. These characteristics provide a high signal to noise ratio andenhance the sensitivity and accuracy of the assay.

[0222] The kit contains two primers that together amplify two ampliconsapproximately 85 base pairs long in region of the CFTR gene thatincludes the F508 region. The sequence of the Limiting primer is 5′CCTGGATTATGCCTGGCACCAT 3′ (SEQ ID NO: 7); it is used at a concentrationof 50 nM. The sequence of the Excess primer is 5′CCTTGATGACGCTTCTGTATCTA 3′(SEQ ID NO: 24); it is used at a concentrationof 1,000 nM. These primers have melting temperatures (T_(m)) that areapproximately matched at the initial concentrations, as calculated usinga nearest neighbor formula (Allawi and SantaLucia, 1997), providingoptimal efficiency and specificity for DNA amplification.

[0223] Materials Provided

[0224] The contents of the CFΔF508 Kit test kit are sufficient toperform an analysis of ten individual samples, each containing 1-10,000cells. All samples, including control samples should be prepared on thesame day. Do not refreeze or reuse any tubes or reagents. Discard unusedmaterials.

[0225] 10 Sample Reaction Tubes containing Cell Lysis Buffer

[0226] 2 No Cell Control Reaction Tubes

[0227] 2 Positive Control Reaction Tubes with DNA heterozygous for ΔF508

[0228] 16 Replacement Caps for Reaction Tubes

[0229] Support Base for Reaction Tubes

[0230] 1 PCR Buffer Tube (235 μl) 60 ml Cell Wash Buffer

[0231] 30 ml Final Wash Buffer

[0232] 12 Sterile transfer pipets

[0233] Form for cell sample identification

[0234] Disposal Bag for used Reaction Tubes

[0235] Additional Materials Required

[0236] inverted microscope or dissecting microscope equipped with micromanipulator or other device for picking up single cells or other smallsamples.

[0237] thermal cycler with fluorescence detection: (ABI PRISM 7700 orequivalent) Laminar flow hood or non-circulating containment hood

[0238] table-top microcentrifuge for 0.2 ml tubes

[0239] micropipets or other device for cell isolation and transfer totubes

[0240] sterile petri dishes

[0241] powder-free gloves

[0242] lab coat, surgical mask and cap

[0243] pipettors and sterile pipet tips with filter

[0244] Platinum Taq DNA Polymerase (Invitrogen) [separate kit module]

[0245] Storage and Handling

[0246] Store the CFΔF508 Kit reagents in a non-frost-free freezer (−20°C.). Avoid repeated thawing and refreezing. Protect the PCR Buffer Tubefrom exposure to light during storage. Handle the tubes and bottles withclean, powder-free gloves.

[0247] General Precautions

[0248] FOR LABORATORY IN VITRO DIAGNOSTIC USE ONLY

[0249] Components of the test must not be used for any purpose otherthan described in these instructions

[0250] Improper handling of the components can lead to contamination andmisdiagnosis of samples

[0251] Improper storage of reagents could affect reactions and preventdiagnosis of samples

[0252] Do not use the enclosed reagents after the expiration date shownon the box

[0253] Contamination Control Precautions

[0254] The CFΔF508 Kit has been designed to minimize the risk ofcontamination. The following steps must be taken to insure that riskremains at an acceptable low level.*Open Reaction Tubes and the PCRBuffer Tube only in a laminar flow hood or non-circulating containmenthood.

[0255] Treat surfaces with 10% bleach or sterilize with UV light priorto use.

[0256] Wear a clean lab coat, surgical mask, cap, and powder-freegloves.

[0257] Handle all kit components ONLY with gloved hands.

[0258] Gloves should be changed after touching any object that might becontaminated with human cells or DNA (e.g., any surface outside thetreated area).

[0259] EXTREME CARE must be taken to avoid transferring unintended cellsto the assay tube. These cells this could provide template for LATE-PCRamplification and Molecular Beacon fluorescence.

[0260] Use mechanical pipetters that are dedicated for PCR setup and arenot used for other purposes.

[0261] Use sterile pipet tips with filters.

[0262] Discard used pipet tips immediately after single use.

[0263] NEVER reintroduce a used pipet tip into a Reaction Tube or thePCR Reagent Tube.

[0264] PCR should be done in a location separate from cell biopsy andsample preparation.

[0265] DO NOT open the Reaction Tubes at anytime after removing themfrom the thermal cycler following PCR amplification. Opening tubesreleases DNA-containing aerosols that can contaminate the laboratory andcould jeopardize all subsequent assays.

[0266] Place used Reaction Tubes in the disposal bag, seal completely,and dispose of properly in accordance with any local, state or federalregulations. Only autoclave waste if required by law If autoclaving isrequired, it should NOT be done in or near the labs used for samplepreparation or PCR.

[0267] CFΔ508 Kit Assay Procedure

[0268] Pre-Biopsy Setup

[0269] STEP A. Place the support base containing 2 No Cell ControlReaction Tubes and 10 Sample Reaction Tubes on ice. After thawing iscomplete, briefly centrifuge all tubes to insure that liquid contentsare at the bottom. Return the tubes to ice, placing them in the properpositions of the support base. Keep all Reaction Tubes closed at thistime.

[0270] STEP B. On the enclosed Cell Identification Form, record thedesignation for each sample to be tested next to the number/color codeof the Sample Reaction Tube that will be used for that embryo. DO NOTplace any marks directly on tubes or caps as this interferes withfluorescence detection.

[0271] STEP C. Working in a containment hood, prepare two petri disheswith Cell Wash Buffer and one petri dish with Final Wash Buffer (0.5 mlto 3.0 ml of buffer per dish) for each embryo to be biopsied. Make surethat each dish is properly labeled.

[0272] Biopsy and Cell Wash: Complete Steps D-F Before Repeating withAnother Embryo.

[0273] STEP D. Care must be taken to avoid transferring non-embryoniccells. In particular in the case of embryo biopsy for PGD, all sperm orcumulus cells surrounding or adhering to the embryo must be removed ascompletely as possible prior to starting the biopsy. Perform embryobiopsy using any established technique, including direct aspiration³,zona drilling and aspiration⁴, zona drilling and displacement⁵, or zonacutting with laser and aspiration⁶. One or two intact blastomeres shouldbe isolated from each embryo. Blastomeres damaged during biopsy orsubsequent wash steps should not be used for diagnosis.

[0274] STEP E1—in cases of embryo biopsy. The following wash steps areimportant to remove components of culture media that can interfere withcell lysis, PCR and fluorescence detection. Transfer the microdropcontaining the blastomere(s) to the hood containing the dissectingmicroscope. While observing under the microscope, use a sterile TransferPipet (provided in the Kit) to move one biopsied blastomere into thefirst dish containing unused Cell Wash Buffer, as follows: A) aspirate asmall amount of Cell Wash Buffer into the transfer pipet; B) aspiratethe blastomere into the tip of the pipet; C) Carefully expel theblastomere into the first dish containing Cell Wash Buffer. As soon asthe blastomere exits the pipet, move the pipet to another region of thedish to expel remaining Cell Wash and rinse the pipet. Repeat thisprocedure to transfer the blastomere into the second dish containingunused Cell Wash Buffer, followed by the third dish containing FinalWash Buffer. All washes should be brief.

[0275] STEP E2—in cases other than human embryos. Cells are placed in a200 μl tube and washed three times in 10 volumes of Cell Wash Buffer bymeans of gentle centrifugation, aspiration of the supernatant, andresuspension in Final Wash Buffer. All washes should be brief and can beperformed at 4° C. if desired.

[0276] Cell Lysis

[0277] STEP F. Use the transfer pipet to pick up the blastomere, orother type of cell, in a small volume of Final Wash Buffer. Theaspirated fluid should not extend more than 1 cm above the tip of thepipet. Open the Sample Reaction Tube that has been designated for thatblastomere and place the cap on a sterile surface. Avoid touching theinner portion of the cap. Place the tip of the transfer pipet containingthe cell directly into the buffer in the Sample Reaction Tube and expelthe contents of the pipet. Close the tube. If bubbles are present, orbuffer droplets are present on the sides of the tube, the sample shouldbe centrifuged briefly (a few seconds). Place the Sample Reaction Tubeon ice as soon as possible after adding the blastomere. Samples MUSTremain on ice until Step I. DO NOT leave the sample at room temperatureas it will result in suboptimal reactions that could prevent accuratediagnosis.

[0278] STEP G. Insure that the correct cell identification has beenrecorded with the Sample Reaction Tube number. If a second cell has beenobtained from the same source, it should be washed and transferred to aseparate Sample Reaction Tube using a new transfer pipet. Discard allused pipets and wash dishes.

[0279] STEP H. Repeat steps D through G using new pipets and wash dishesfor each embryo tested. Transfer an equivalent volume of Final WashBuffer into both of the No Cell Control Reaction Tubes.

[0280] STEP I. Preheat the thermal cycler block to 50° C. The block mustbe equipped with a heated cover designed to prevent condensation on thelid of the tubes. Place all Sample Reaction Tubes, as well as both NoCell Control Reaction Tubes into the preheated block. Incubate at 50° C.for 30 min, then 95° C. for 15 minutes. Once the block has cooled toroom temperature, remove and examine each Reaction Tube. If condensationis present on the cap or sides of tubes, the samples may not provideadequate amplification for diagnosis. Place tubes on ice as described inStep A. During the incubation period of step I, continue with Steps Jand K.

[0281] PCR Setup (Steps K and L Should Either be Carried Out in aDifferent Laminar Flow Hood or Non-Circulating Containment Hood thanthat Used for Steps D-H, or the Hood Used for Steps D-H Should beCleared of all Unnecessary Materials and Wiped Down with a 10% BleachSolution Prior to Using it for Steps K and L)

[0282] STEP J. [LATE-PCR with Hotstart]

[0283] Add 4.8 μl of Platinum Taq DNA Polymerase (5 Units/μl) to the PCRBuffer Tube and mix thoroughly to insure even distribution of theenzyme.

[0284] STEP K. Thaw the two Normal/ΔF508 heterozygous DNA Control Tubesby placing them in the support rack on ice. Add each Sample ReactionTube and each No Cell Control Reaction Tube to the support rack once thelysis incubation (step J) is complete.

[0285] STEP L. Open the first Sample Reaction Tube and add 15 μl of PCRBuffer containing added Taq DNA polymerase. Recap the tube immediatelyusing a Replacement Cap. Discard the old cap and used pipet tip. Repeatthis step for each Reaction Tube, including the two Positive ControlTubes and the two No Cell Control Tubes. All Control Tubes must beanalyzed in parallel with unknown cell samples for accurateinterpretation of test results. As an added precaution against crosscontamination, it is recommended that gloves be changed after eachsample.

[0286] PCR and Fluorescence Detection

[0287] STEP M. [Recommended Cycling Parameters]

[0288] Load the tubes into a thermal cycler with fluorescence detection.The following program is based on the ABI PRISM 7700 Sequence Detector(Applied Biosystems):

[0289] Stage 1. 95° C., 3 minutes (1 repeat)

[0290] Stage 2. 95° C., 10 seconds, 55° C. 30 seconds, 72° C. 30 seconds(25 repeats)

[0291] Stage 3. 95° C., 10 seconds, 52° C. 30 seconds, 72° C. 30 seconds(35 repeats)

[0292] Set fluorescence acquisition for FAM and TET during the 52° C.step.

[0293] Set sample volume at 25 μl.

[0294] Select the block positions that are used and type in the correctsample identifications.

[0295] STEP N. Never open the Reaction Tubes following amptification asthis may result in contamination of the laboratory that could jeopardizeall subsequent assays. Following the completion of PCR, remove allReaction Tubes without opening and immediately place them in thedisposal bag. Completely seal the bag and dispose of properly inaccordance with any local, state or federal regulations. If autoclavingis required, seal the bag with as little air as is reasonably possible,then proceed to autoclave at a site distant from the laboratory, in anautoclave that is not used to prepare any other materials used in thelaboratory.

[0296] Analysis and Interpretation of Assay Results

[0297] The following description is appropriate for an ABI PRISM 7700Sequence Detector (Applied Biosystems); parameters appropriate for othermachines are currently unknown. Set a threshold value of 200 units and abaseline start at cycle 3 and stop at cycle 12 of stage 3 (cycles 28 and37 of the overall reaction) for each reporter dye (FAM and TET) in theanalysis window of the ABI 7700. These values are used to compute thethreshold cycle (C_(T)) for fluorescence detection in each sample.

[0298] The range of C_(T) values and Final Fluorescence values requiredfor scoring positive for the normal allele (FAM signal) and for theΔF508 mutant allele (TET signal) are provided on the separate ProductAnalysis Certificate and are based on testing for each lot. Values abovethreshold, but below these required values indicate the possibility ofcontamination or inadequate cell lysis. Samples generating such valuesshould not be scored and embryos should not be transferred based on thetest results.

[0299] Limitations of the Test

[0300] The CFAF508 Kit has been designed to use Platinum Taq DNAPolymerase (Invitrogen) and the ABI PRISM 7700 Sequence Detector(Applied Biosystems). Use of other DNA polymerase with hot start orother thermal cyclers with fluorescence detection may be possible, butmust be optimized by the user prior to testing cells from embryos. Thiskit is designed to identify the ΔF508 mutation and the normal allelesequence in that region of the CFTR gene. Other mutations in the CFTRgene cannot be detected using this kit.

[0301] Chromosomal mosaicism is present in some embryos obtained fromIVF and is common in embryos with poor morphology. A single biopsiedblastomere therefore may not be representative of the genetic makeup ofthe embryo.

[0302] Some human embryos contain a nucleate cells that will givenegative results if used in this assay. The most accurate results willbe obtained if nuclei are observed in biopsied blastomeres. A blastomereshould not be used if signs of cell lysis are observed following biopsy,since cell damage may include DNA degradation.

[0303] References

[0304]¹Tyagi, S. and Kramer, F. R. (1996) Nature Biotechnology 14,303-308

[0305]²Piatek et al. (1998) Nature Biotechnology 16, 359-363

[0306]³Wilton, L. J. and Trounson, A. O. (1989) Biology of Reproduction40, 145-152

[0307]⁴Hardy, K. et al. (1990) Human Reproduction 5, 708-714

[0308]⁵Pierce, K. E. et al. (1997) Human Reproduction 12, 351-356

[0309]⁶Boada et al. (1998) J. Assist Reprod Genet 15, 302-307

[0310] CFA508 Kit Components and Compositions

[0311] Reaction Tubes

[0312] 0.2 ml Optical Tubes from Perkin Elmer (Pt # N801-0933)

[0313] with optical caps (Pt # N801-0935)

[0314] Composition of Cell Lysis Buffer (Sample and No Cell ReactionTubes):

[0315] 100 μg/ml proteinase K (Roche Molecular Biochemicals)

[0316] 5 μM Sodium Dodecylsulfate

[0317] 10 mM Tris, pH 8.3 (Trizma pre-set crystals, Sigma)

[0318] molecular-grade water

[0319] Composition of Sample Contained in Normal/Δ508 Control ReactionTube:

[0320]60 picograms human DNA heterozygous for the Δ508 allele of the CFgene.

[0321] 5 μM Sodium Dodecylsulfate

[0322] 10 mM Tris, pH 8.3 (Trizma pre-set crystals, Sigma)

[0323] molecular-grade water to 10 microliter final volume

[0324] Composition of Cell Wash Buffer:

[0325] Phosphate buffered saline without Ca⁺⁺ or Mg⁺⁺ (Sigma, Cat. No.D-8537)

[0326] 0.1% Polyvinylpyrrolidone (Sigma, Cat. No. PVP-40)

[0327] Composition of Final Wash Buffer:

[0328] Phosphate buffered saline without calcium or magnesium

[0329] 0.01% Polyvinylpyrrolidone

[0330] Composition of PCR Buffer:

[0331] 1.67×PCR Buffer (Invitrogen)

[0332] 6.25 mM MgCl₂

[0333] 0.42 mM each of the four deoxyribonucleotide triphosphates (dCTP,dTTP, DATP, dGTP)

[0334] 0.083 μM Limiting Primer

[0335] 1.67 μM Excess Primer

[0336] 1.0 μM each Molecular Beacon (2 total)

[0337] Molecular-grade water to final volume of 235 microliters

[0338] CF Primers:

[0339] Limiting: 5′ CCTGGATTATGCCTGGCACCAT 3′ [SEQ ID NO: 7]

[0340] Excess: 5′ CCTTGATGACGCTTCTGTATCTA 3′ [SEQ ID NO: 24]

[0341] Normal Allele Molecular Beacon:

[0342] 5′ FAM-CGCGCTTATCATCTTTGGTGTTTCCTATAGCGCG-Dabcyl 3′ [SEQ ID NO:9]

[0343] Δ508 Allele Molecular Beacon:

[0344] 5′ TET-CGCGCTAAAATATCATTGGTGTTTCCTAAGCGCG-Dabcyl 3′ [SEQ ID NO:23]

1 24 1 20 DNA Artificial Sequence primer 1 ccttctctct gccccctggt 20 2 22DNA Artificial Sequence primer 2 gccaggggtt ccactacgta ga 22 3 22 DNAArtificial Sequence primer 3 gcccttctct ctgccccctg gt 22 4 18 DNAArtificial Sequence primer 4 gattatgcct ggcaccat 18 5 23 DNA ArtificialSequence primer 5 ctttgatgac gcttctgtat cta 23 6 19 DNA ArtificialSequence primer 6 ggattatgcc tggcaccat 19 7 22 DNA Artificial Sequenceprimer 7 cctggattat gcctggcacc at 22 8 24 DNA Artificial Sequence primer8 ccgcccttct ctctgccccc tggt 24 9 34 DNA Artificial Sequence probe 9cgcgcttatc atctttggtg tttcctatag cgcg 34 10 19 DNA Artificial Sequenceprimer 10 tgattatgcc tggcaccat 19 11 21 DNA Artificial Sequence primer11 cttgattatg cctggcacca t 21 12 20 DNA Artificial Sequence primer 12tggattatgc ctggcaccat 20 13 21 DNA Artificial Sequence primer 13ctggattatg cctggcacca t 21 14 23 DNA Artificial Sequence primer 14tcctggatta tgcctggcac cat 23 15 29 DNA Artificial Sequence primer 15cagttttcct ggattatgcc tggcaccat 29 16 30 DNA Artificial Sequence primer16 tcagttttcc tggattatgc ctggcaccat 30 17 16 DNA Artificial Sequenceprimer 17 gacgcttctg tatcta 16 18 17 DNA Artificial Sequence primer 18tgacgcttct gtatcta 17 19 18 DNA Artificial Sequence primer 19 atgacgcttctgtatcta 18 20 20 DNA Artificial Sequence primer 20 tgatgacgcttctgtatcta 20 21 24 DNA Artificial Sequence primer 21 gctttgatgacgcttctgta tcta 24 22 29 DNA Artificial Sequence primer 22 ggcatgctttgatgacgctt ctgtatcta 29 23 34 DNA Artificial Sequence probe 23cgcgctaaaa tatcattggt gtttcctaag cgcg 34 24 23 DNA Artificial Sequenceprimer 24 ccttgatgac gcttctgtat cta 23

What is claimed is:
 1. A non-symmetric polymerase chain reaction (PCR)amplification method comprising thermally cycling a PCR reaction mixturecontaining a deoxyribonucleic acid (DNA) amplification target sequence,a pair of PCR primers, dNTP's and a thermostable polymerase repeatedlythrough PCR steps of strand melting, primer annealing and primerextension, wherein, at the outset of amplification (a) the reactionmixture contains up to 1,000,000 copies of the amplification targetsequence, (b) the PCR primer pair comprises a limiting primer and anexcess primer, the limiting primer being present at a concentration ofup to 200 nM and the excess primer being present at a concentration atleast five times higher than the limiting primer, (c) the initial,concentration-adjusted melting temperature of the limiting primer isequal to or greater ii than the initial, concentration-adjusted meltingtemperature of the excess primer, (d) if the limiting primer is notfully complementary to said target sequences the concentration-adjustedmelting temperature of that portion of the limiting primer whichhybridizes to said target sequence is not more than 5° C. below theconcentration-adjusted melting temperature of the excess primer, (e) themelting temperature of the amplicon produced by extension of the excessprimer exceeds the initial concentration-adjusted melting temperature ofthe excess primer by not more than 18° C., and (f) thermal cycling isrepeated a number of times sufficient to include multiple cycles oflinear amplification using the excess primer following exhaustion of thelimiting primer.
 2. The method of claim 1 for at least two targetsequences wherein the reaction mixture includes a pair of PCR primersfor each target, and wherein the initial, concentration-adjusted meltingpoints of all limiting primers are equal to or greater than the initial,concentration-adjusted melting points of all excess primers.
 3. Themethod of claim 1 further comprising reverse transcribing a ribonucleicacid (RNA) molecule to generate said DNA target sequence.
 4. The methodof claim 1 wherein the reaction mixture contains up to 50,000 copies ofthe nucleic acid target.
 5. The method of claim 1 wherein the initial,concentration-adjusted melting temperature of the limiting primer is atleast 3° C. higher than the initial, concentration-adjusted meltingtemperature of the excess primer.
 6. The method of claim 5 wherein theexcess primer is present at a concentration of 500-2000 nM and at leastten times higher than the limiting primer.
 7. The method of claim 5wherein the melting temperature of the amplicon is 7-15° C. higher thanthe initial, concentration-adjusted melting temperature of the excessprimer.
 8. The method of claim 4 wherein the melting temperature of theamplicon is 7-15° C. higher than the initial, concentration-adjustedmelting temperature of the excess primer.
 9. The method of claim 4wherein the initial, concentration-adjusted melting temperature of thelimiting primer is at least 3° C. higher than the initial,concentration-adjusted melting temperature of the excess primer.
 10. Themethod of claim 1 wherein the melting temperature of the amplicon is7-15° C. higher than the initial, concentration-adjusted meltingtemperature of the excess primer.
 11. The method of claim 1 wherein thereaction mixture contains up to 1000 copies of the DNA target.
 12. Themethod of claim 11 wherein the initial, concentration-adjusted meltingtemperature of the limiting primer is at least 3° C. higher than theinitial, concentration-adjusted melting temperature of the excessprimer.
 13. The method of claim 11 wherein the melting temperature ofthe amplicon is 7-15° C. higher than the initial, concentration-adjustedmelting temperature of the excess primer.
 14. The method of claim 1wherein the duration of the primer annealing step is not longer than 30seconds.
 15. The method according to claim 1 further including at leastone terminal thermal cycle in which the single-stranded extensionproduct of the excess primer is converted to double-stranded product,wherein the PCR reaction mixture additionally includes a low-temperatureprimer capable of priming the extension product of the excess primer andhaving an initial, concentration-adjusted melting temperature at least5° C. below the initial, concentration-adjusted melting temperature ofthe excess primer, and wherein the annealing temperature is maintainedabove the initial, concentration-adjusted melting temperature of thelow-temperature primer except for at least one terminal cycle in whichthe annealing temperature is lowered to hybridize the low-temperatureprimer.
 16. The method according to claim 15 wherein the initial,concentration-adjusted melting temperature of the low-temperature primeris at least 10° C. below the initial, concentration-adjusted meltingpoint of the excess primer.
 17. The method of claim 1 wherein thereaction mixture additionally contains a complementary oligonucleotidethat hybridizes to the 3′ end of the excess primer and has an initial,concentration-adjusted melting temperature at least 3° C. lower than theinitial, concentration-adjusted melting temperature of the excessprimer.
 18. The method of claim 17 wherein the complementaryoligonucleotide is present at the outset in a concentration greater thanthe concentration of the excess primer.
 19. A non-symmetric polymerasechain reaction (PCR) method comprising thermally cycling a PCR reactionmixture containing a deoxyribonucleic acid (DNA) amplification targetsequence, a pair of matched limiting PCR primers, an additional excessprimer, dNTP's and a thermostable polymerase repeatedly through PCRsteps of strand melting, primer annealing and primer extension, whereinthe matched PCR primers are present in approximately equimolarconcentration of up to 200 nM, the excess primer is present at aconcentration at least five times higher than the limiting primers, theinitial, concentration-adjusted melting temperatures of the excessprimer is at least 5° C. below the initial, concentration-adjustedmelting temperature of the limiting primers, and wherein the reactioncomprises a first phase wherein the annealing temperature is higher thanthe initial, concentration-adjusted melting temperature of the excessprimer and the matched limiting primers generate a first amplicon, and asecond phase wherein the annealing temperature is lowered and the excessprimer generates a second amplicon, shorter than the first amplicon,utilizing the first amplicon as a template strand, and wherein themelting temperature of the second amplicon exceeds the initial,concentration-adjusted melting temperature of the excess primer by notmore than 25° C.
 20. The method according to claim 19, wherein theinitial concentration-adjusted melting temperature of the excess primeris at least 10° C. below the initial, concentration-adjusted meltingtemperatures of the limiting primers.
 21. The method according to claim20 wherein the melting temperature of the second amplicon exceeds theinitial, concentration-adjusted melting temperature of the excess primerby not more than 18° C.
 22. A non-symmetric polymerase chain reaction(PCR) method with removal of single-stranded amplicon comprising a)thermally cycling a PCR reaction mixture containing a DNA amplificationtarget sequence, a pair of PCR primers for said amplification targetsequence, dNTP's and a thermostable DNA polymerase through repeatedcycles of strand melting, primer annealing and primer extension, wherein(i) the PCR primer pair comprises a limiting primer and an excessprimer, (ii) the limiting primer is present at a concentration of up to200 nM, and the excess primer is present at a concentration at leastfive times higher than the limiting primer, (iii) the initial,concentration-adjusted melting temperature of the limiting primer is atleast equal to the initial, concentration-adjusted melting temperatureof the excess primer, and (iv) thermal cycling is repeated a number oftimes sufficient to include multiple cycles of linear amplificationusing the excess primer following exhaustion of the limiting primer; andb) during at least some cycles of linear amplification, following thestep of primer extension, removing single-stranded extension product ofthe excess primer from the reaction mixture by hybridizing said productto capture probes.
 23. The method of claim 22 wherein said captureprobes are in a thermally isolated product removal zone and said step ofremoving comprises passing the reaction mixture through said zone. 24.The method according to claim 23 wherein said product removal zone isphysically isolatable from said at least one reaction zone, furtherincluding periodically isolating said product removal zone andharvesting product hybridized to said capture probes while the reactionmixture is in said at least one reaction zone.
 25. The method of claim22 wherein the initial, concentration-adjusted melting temperature ofthe limiting primer is at least 3° C. higher than the initial,concentration-adjusted melting temperature of the excess primer.
 26. Themethod of claim 22 wherein the melting temperature of the ampliconexceeds the initial concentration-adjusted melting temperature of theexcess primer by not more than 18° C.
 27. The method of claim 22 whereinthe excess primer is present at a concentration of 500-2000 nM and atleast ten times higher than the limiting primer.
 28. A homogeneousdetection assay for at least one DNA amplification target sequenceemploying non-symmetric polymerase chain reaction (PCR) amplification,comprising (a) thermally cycling through multiple cycles of PCR steps ofstrand melting, primer annealing and primer extension a PCR reactionmixture containing said at least one amplification target sequence, athermostable DNA polymerase, dNTP's, and for each amplification targetsequence a pair of PCR primers for amplifying said amplification targetsequence and at least one labeled hybridization probe that hybridizes tothe amplicon produced by said primers and (b) detecting signal producedby said at least one probe as an indication of the presence of said atleast one amplification target sequence, wherein (i) each PCR primerpair comprises a limiting primer and an excess primer, (ii) the limitingprimer is present at a concentration of up to 200 nM, and for eachprimer pair the excess primer is present at a concentration of at leastfive times higher than the limiting primer concentration, (iii) theinitial, concentration-adjusted melting temperatures of all limitingprimers are at least equal to the initial, concentration-adjustedmelting temperatures of all excess primers (iv) for each primer pair themelting temperature of the amplicon exceeds the initial,concentration-adjusted melting temperature of the excess primer by notmore than 25° C., (v) thermal cycling is repeated a number of timessufficient to include multiple cycles of linear amplification using theexcess primers following exhaustion of the limiting primers, and (vi)said probes are selected from the group consisting of probes thathybridize to the extension product of the limiting primer, and probesthat hybridize to the extension product of the excess primer and signalupon hybridization, and (viii) said probes hybridize to said ampliconsduring the PCR step of primer annealing.
 29. The assay according toclaim 28 where said step of detecting is an end-point detection.
 30. Theassay according to claim 28 wherein said step of detection is real-timedetection is performed during the annealing step of at least some cyclesof linear amplification.
 31. The assay according to claim 28 whereinsaid step of detection is real-time detection performed following theextension step of at least some cycles of linear amplification and priorto strand melting of the following cycles.
 32. The assay according toclaim 31 wherein the duration of the primer annealing step is not longerthan 30 seconds.
 33. The assay according to claim 32 wherein, for eachprimer pair, the melting temperature of the amplicon exceeds theinitial, concentration-adjusted melting temperature of the excess primerby not more than 18° C.
 34. The assay according to claim 32 wherein, foreach primer pair, the initial, concentration-adjusted meltingtemperature of the limiting primer is at least 3° C. higher than theinitial, concentration-adjusting melting temperature of the excessprimer.
 35. The assay according to claim 32 wherein said probeshybridize to the extension products of the excess primers and signalupon hybridization.
 36. The assay according to claim 28 wherein said atleast one hybridization probe is a dual-labeled fluorescent probe thathybridizes to the extension product of the limiting primer and that ishydrolyzed by the polymerase during extension of the excess primer,thereby generating a detectable signal, and wherein said signal isdetected in real time during at least some cycles of said linearamplification.
 37. The assay according to claim 36, wherein the reactionmixture contains up to 50,000 of the at least one amplification targetsequence.
 38. The assay according to claim 37 wherein, for each primerpair, the melting temperature of the amplicon is not more than 18° C.higher than the initial, concentration-adjusted melting temperature ofthe excess primer.
 39. The assay according to claim 37 wherein, for eachprimer pair, the initial, concentration-adjusted melting temperature ofthe limiting primer is at least 3° C. higher than the initial,concentration-adjusted melting temperature of the excess primer.
 40. Theassay according to claim 39 wherein the duration of the primer annealingstep is not longer than 30 seconds.
 41. The assay according to claim 37,wherein said signal from said at least one hybridization probe isdetected between the PCR steps of primer extension and strand melting.42. The assay according to claim 28 wherein said at least onehybridization probe is a dual-labeled fluorescent probe that hybridizesto the extension product of the limiting primer and that is hydrolyzedby the polymerase during extension of the excess primer, therebygenerating a detectable signal.
 43. The assay according to claim 28wherein said at least one hybridization probe emits a detectable signalupon hybridization to the extension product of the excess primer. 44.The assay according to claim 43 wherein, for at least one PCR primerpair, the reaction mixture additionally contains a complementaryoligonucleotide that hybridizes to the 3′ end of the excess primer andhas an initial, concentration-adjusted melting temperature at least 3°C. lower than the initial, concentration-adjusted melting temperature ofthe excess primer.
 45. The assay according to claim 44 wherein thecomplementary oligonucleotide is present at the outset in aconcentration greater than the concentration of the excess primer. 46.The assay according to claim 28 wherein said at least one hybridizationprobe comprises a first probe for one allelic variant and a second probefor another allelic variant.
 47. The assay according to claim 28 furthercomprising reverse transcribing of ribonucleic acid (RNA) molecule togenerate said DNA target sequence.
 48. The assay according to claim 28wherein the reaction mixture contains up to 50,000 copies of the nucleicacid target.
 49. The assay according to claim 28 wherein the initial,concentration-adjusted melting temperature of the limiting primer is atleast 3° C. higher than the in initial, concentration-adjusted meltingtemperature of the excess primer.
 50. The assay according to claim 28wherein the melting temperature of the amplicon is not more than 18° C.higher than the initial, concentration-adjusted melting temperature ofthe excess primer.
 51. The assay according to claim 28 wherein said atleast one amplification target sequence comprises at least twoamplification target sequences.
 52. A homogeneous detection assay for atleast one DNA amplification target sequence employing non-symmetricpolymerase chain reaction (PCR) amplification, comprising (a) thermallycycling through multiple cycles of PCR steps of strand melting, primerannealing and primer extension a PCR reaction mixture containing said atleast one amplification target sequence, a thermostable DNA polymerase,dNTP's, and for each amplification target sequence a pair of PCR primersfor amplifying said amplification target sequence and at least onelabeled low-temperature hybridization probe that hybridizes to theamplicon produced by said primers and (b) detecting signal produced bysaid at least one probe as an indication of the presence of said atleast one amplification target sequence, wherein (i) each PCR primerpair comprises a limiting primer and an excess primer, (ii) eachlimiting primer is present at a concentration of up to 200 nM, and foreach primer pair the excess primer is present at a concentration atleast five times higher than the limiting primer concentration, (iii)for each amplification target sequence, the low-temperaturehybridization probe binds to the extension product of the excess primerand emits a detectable signal upon hybridization, (iv) for eachamplification target sequence the initial, concentration-adjustedmelting temperature of the low-temperature hybridization probe is atleast 5° C. below the initial, concentration-adjusted meltingtemperature of the limiting primer, (v) thermal cycling is repeated anumber of times sufficient to include multiple cycles of linearamplification using the excess primers following exhaustion of thelimiting primers, and (vi) detection is performed at a temperaturesufficiently low for said low-temperature probes to hybridize andsignal.
 53. The assay according to claim 52, wherein the initial,concentration-adjusted melting temperature of the low-temperaturehybridization probe is at least 10° C. below the initial,concentration-adjusted melting temperature of the limiting primer. 54.The assay according to claim 52 wherein each probe is present in saidreaction mixture at a concentration of at least 1 uM.
 55. The assayaccording to claim 52 wherein the PCR step of primer annealing during atleast some cycles of linear amplification is of sufficiently lowtemperature and of sufficient duration that the low-temperature probehybridizes during primer annealing, and detection is performed duringthat step.
 56. The assay according to claim 52 wherein the initial,concentration-adjusted melting temperatures of all limiting primers areat least equal to the initial, concentration-adjusted meltingtemperatures of all excess primers.
 57. The assay according to claim 52wherein the PCR amplification includes an added detection step followingprimer extension during at least some cycles of linear amplification,said detection step being of sufficiently low temperature and sufficientduration for the low-temperature hybridization probes to hybridize andsignal, and wherein the PCR step of primer annealing is not ofsufficiently low temperature and sufficient duration for said probes tohybridize and signal.
 58. The assay according to claim 57, wherein theinitial, concentration-adjusted melting temperature of thelow-temperature hybridization probe is at least 10° C. below theinitial, concentration-adjusted melting temperature of the limitingprimer
 59. The assay according to claim 57, wherein the initial,concentration-adjusted melting temperature of the low-temperaturehybridization probe is at least 5° C. below the mean temperature of theannealing step of the exponential phase of the amplification reaction.60. The assay according to claim 59, wherein the initial,concentration-adjusted melting temperature of the low-temperaturehybridization probe is at least 10° C. below the mean temperature of theannealing step of the exponential phase of the amplification reaction.61. The assay according to claim 57, wherein the detection step isperformed only beginning a few cycles prior to the threshold cycle ofthe reaction.
 62. The assay according to claim 57, wherein thelow-temperature detection step is not more than 30 seconds duration. 63.The assay according to claim 57, wherein detection is end-pointdetection performed following completion of the amplification reaction.64. The assay according to claim 52, wherein for each primer pair themelting temperature of the amplicon exceeds the initial,concentration-adjusted melting temperature of the excess primer by notmore than 25° C.
 65. A method for amplification of a nucleic acid targetsequence present in a sample containing up to about 10,000 copies ofsaid target sequence, the method comprising: a) contacting the nucleicacid target sequence with a first oligonucleotide primer and a secondoligonucleotide primer, wherein the T_(m) of the first primer is atleast 5° C. greater than the T_(m) of the second primer and wherein theconcentration of the second primer is up to 1000 nM and at least about10 times greater than the concentration of the first primer; and b)amplifying the target sequence by a polymerase chain reaction utilizingsaid first and second oligonucleotide primers, said reaction having anexponential phase of amplicon generation followed by a linear phase ofamplicon generation that utilizes only the second primer.
 66. The methodaccording to claim 65 wherein said sample contains nucleic acid from asingle cell.
 67. The method according to claim 65 wherein the T_(m) ofthe first primer is 10° C.-20° C. greater than the T_(m) of the secondprimer.
 68. The method according to claim 67 wherein the concentrationof the second primer is 20-100 times greater than the concentration ofthe first primer.
 69. The method according to claim 65 wherein theconcentration of the second primer is 20-100 times greater than theconcentration of the first primer.
 70. A method for detecting at leastone nucleic acid target sequence in a sample containing up to about10,000 copies of said at least one target sequence, the methodcomprising: a) contacting the at least one nucleic acid target sequencewith a first oligonucleotide primer hybridizable thereto and a secondoligonucleotide primer hybridizable thereto, wherein the T_(m) of thefirst primer is at least 5° C. greater than the T_(m) of the secondprimer and wherein the concentration of the second primer is up to 1000nM and at least about 10 times greater than the concentration of thesecond primer; b) amplifying the at least one target sequence by apolymerase chain reaction utilizing said first and secondoligonucleotide primers, said reaction having an exponential phase ofamplicon generation followed by a linear phase of amplicon generationthat utilizes only the ii second primer; and c) detecting amplicongenerated from said second primer in real time during the polymerasechain reaction by means of a first hybridization probe targeted thereto.71. The method according to claim 70 wherein said at least one nucleicacid sequence comprises a genetic sequence subject to allelic mutationwherein said first hybridization probe is targeted to a first allelicvariant.
 72. The method according to claim 71 further comprisingdetecting amplicon generated from said second primer in real time duringthe polymerase chain reaction by means of a second hybridization probetargeted to a second allelic variant.
 73. The method according to claim71 wherein the T_(m) of the first primer is 10° C.-20° C. greater thanthe T_(m) of the second primer.
 74. The method according to claim 71wherein the T_(m) of the first primer is 10° C.-20° C. greater than theT_(m) of the second primer.
 75. The method according to claim 71 whereinthe concentration of the second primer is 20-100 times the concentrationof the first primer.
 76. The method according to claim 71 wherein theconcentration of the second primer is 20-100 times the concentration ofthe first primer.
 77. The method according to claim 70 wherein said atleast one nucleic acid sequence comprises at least two different nucleicacid sequences, and wherein the method comprises contacting each nucleicacid sequence with a first primer hybridizable thereto and a secondprimer hybridizable thereto.
 78. The method according to claim 70wherein the concentration of the second primer is 20-100 times theconcentration of the first primer.
 79. The method according to claim 70wherein the T_(m) of the first primer is 10° C.-20° C. greater than theT_(m) of the second primer.
 80. The method according to claim 79 whereinthe concentration of the second primer is 20-100 times the concentrationof the first primer. 81 The method according to claim 70, whereindetection is performed between the PCR steps of primer extension andstrand melting.
 82. The method according to claim 81, wherein thedetection is performed at a temperature below the primer extensiontemperature.
 83. The method according to claim 81 wherein said firsthybridization probe is a molecular beacon probe.
 84. An oligonucleotideset comprising a pair of primers for amplifying a selected DNA sequenceby a non-symmetric polymerase chain reaction (PCR) comprising a firstamount of a limiting primer intended to be used at a concentration ofnot more than 200 nM and a second amount, at least five times greaterthan said first amount, of an excess primer intended to be used at aconcentration at least five times greater than the limiting primerconcentration, wherein the intended, concentration-adjusted meltingpoint of the limiting primer is equal to or greater than the intendedconcentration-adjusted melting point of the excess primer, and whereinthe melting point of the amplicon produced by said primer pair does notexceed the intended concentration-adjusted melting point of the excessprimer by more than 18° C.
 85. The oligonucleotide set according toclaim 84 wherein the intended concentration-adjusted melting point ofthe limiting primer is at least 3° C. higher than the intendedconcentration-adjusted melting point of the excess primer.
 86. Theoligonucleotide set according to claim 84 wherein said second amount isat least ten times greater than said first amount and wherein saidexcess primer is intended to be used at a concentration at least tentimes greater than the limiting primer concentration.
 87. Theoligonucleotide set according to claim 86 wherein the melting point ofthe amplicon exceeds the intended concentration-adjusted melting pointof the excess primer by 7-15° C.
 88. The oligonucleotide set accordingto claim 85 wherein the melting point of the amplicon exceeds theintended concentration-adjusted melting point of the excess primer by7-15° C.
 89. The oligonucleotide set according to claim 84 wherein themelting point of the amplicon exceeds the intendedconcentration-adjusted melting point of the excess primer by 7-15° C.90. An oligonucleotide set for amplifying a selected DNA sequence by apolymerase chain reaction (PCR) comprising a pair of matched PCRlimiting primers for producing a first amplicon and an excess primer forproducing a second, single-stranded amplicon utilizing said firstamplicon as a template, wherein (i) each of said limiting primers ispresent in a first amount and is intended to be used at a concentrationnot exceeding 200 nM, (i) said excess primer is present in a secondamount, at least five times greater than said first amount, and isintended to be used at a concentration at least five times higher thanthe concentration of each limiting primer, and (iii) wherein theintended concentration-adjusted melting temperature of the excess primeris at least 5° C. lower than the intended concentration-adjusted meltingtemperature of both limiting primers.
 91. The oligonucleotide setaccording to claim 90 wherein the melting temperature of the secondamplicon exceeds the intended concentration-adjusted melting temperatureof the excess primer by not more than 18° C.
 92. The oligonucleotide setaccording to claim 90 wherein said second amount is at least ten timesgreater than said first amount and wherein said excess primer isintended to be used at a concentration at least ten times greater thanthe limiting primer concentration.
 93. An oligonucleotide set for ahomogenous, non-symmetric polymerase chain reaction (PCR) amplificationassay for a selected DNA sequence comprising a first amount of alimiting primer intended to be used at a concentration not exceeding 200nM, a second amount, at least five times greater than said first amount,of an excess primer intended to be used at a concentration at least fivetimes greater than the limiting primer concentration, and a labeledhybridization probe that emits a signal indicative of the presence ofproduct produced by extension of said excess primer, wherein theintended concentration-adjusted melting temperature of the limitingprimer is at least equal to the intended concentration-adjusted meltingtemperature of the excess primer, and wherein the melting point of theamplicon exceeds the intended concentration-adjusted melting point ofthe excess primer by not more than 25° C.
 94. The oligonucleotide setaccording to claim 93 wherein the melting temperature of the ampliconexceeds the intended concentration-adjusted melting temperature of theexcess primer by not more than 18° C.
 95. The oligonucleotide setaccording to claim 93, wherein the intended concentration-adjustedmelting temperature of the limiting primer exceeds the intendedconcentration-adjusted melting temperature of the excess primer by atleast 3° C.
 96. The oligonucleotide set according to claim 93, whereinsaid second amount is at least ten times greater than said first amountand wherein said excess primer is intended to be used at a concentrationat least ten times greater than the limiting primer concentration. 97.The oligonucleotide set according to claim 93, wherein said probehybridizes to the extension product of the excess primer and signalsupon hybridization.
 98. The oligonucleotide set according to claim 97,wherein said probe comprises a first hybridization probe specific for afirst allele and a second hybridization probe specific for a secondallele.
 99. The oligonucleotide set according to claim 97, wherein saidhybridization probe is a molecular beacon probe.
 100. Theoligonucleotide set according to claim 97, wherein said probe isintended to be used at an intended probe concentration and wherein theintended probe concentration-adjusted melting point of the probe is atleast 5° C. below the intended concentration-adjusted melting point ofthe limiting primer.
 101. The oligonucleotide set according to claim100, wherein the intended probe concentration-adjusted melting point ofthe probe is at least 10° C. below the intended concentration-adjustedmelting point of the limiting primer.
 102. The oligonucleotide setaccording to claim 101, wherein said probe is a molecular beacon probe.103. A kit of reagents for performing a homogeneous polymerase chainreaction assay for at least one pre-selected DNA target sequence,comprising at least one pair of polymerase chain reaction primersincluding a first primer and a second primer, four deoxyribonucleotidetriphosphates, a thermostable DNA polymerase, and a labeledhybridization probe that emits a detectable signal upon hybridization,wherein a) the T_(m) of the first primer is at least 5° C. greater thanthe T_(m) of the second primer and the concentration of the secondprimer is at least ten times greater than the concentration of the firstprimer, and b) said labeled hybridization probe binds to the extensionproduct of said second primer.
 104. The kit according to claim 103wherein the T_(m) of the first primer is 10° C.-20° C. greater than theT_(m) of the second primer.
 105. The kit according to claim 104 whereinthe concentration of the second primer is 20-100 times greater than theconcentration of the first primer.
 106. The kit according to claim 103wherein the concentration of the second primer is 20-100 times greaterthan the concentration of the first primer.
 107. A kit of reagents forperforming a homogenous polymerase chain reaction (PCR) assay for atleast one pre-selected DNA target sequence comprising a thermostable DNApolymerase, dNTP's and, for each target sequence, a first amount of alimiting primer to be used at a concentration of up to 200 nM, a secondamount, at least five times greater than said first amount, of an excessprimer to be used at a concentration at least give times greater thanthe limiting primer concentration, and a labeled hybridization probethat emits a signal indicative of the presence of product produced byextension of said excess primer, wherein the initial,concentration-adjusted melting temperature of the limiting primer is atleast equal to the initial concentration-adjusted melting temperature ofthe excess primer, and wherein the melting temperature of the ampliconexceeds the initial concentration-adjusted melting temperature of theexcess primer by not more than 25° C.
 108. The kit according to claim107 for performing a multiplex assay for at least two target sequences,wherein the initial, concentration-adjusted melting temperatures of alllimiting primers are at least equal to the initialconcentration-adjusted melting temperatures of all excess primers. 109.The kit according to claim 107, wherein the initialconcentration-adjusted melting temperature of the limiting primer is atleast 3° C. higher than the initial, concentration-adjusted meltingtemperature of the excess primer.
 110. The kit according to claim 107,wherein said probe hybridizes to the extension product of the excessprimer and signals upon hybridization.
 111. The kit according to claim110, wherein said probe for said at least one target comprises a firstprobe for one allelic variant and a second probe for a second allelicvariant.
 112. The kit according to claim 110 for performing a multiplexassay for at least two target sequences, wherein the initial,concentration-adjusted melting temperatures of all limiting primers areat least equal to the initial concentration-adjusted meltingtemperatures of all excess primers.
 113. The kit according of claim 110,wherein the initial, concentration-adjusted melting temperature of saidlimiting primer is at least 3° C. higher than the initial,concentration-adjusted melting temperature of said excess primer. 114.The kit according to claim 110 wherein the initial,concentration-adjusted melting temperature of said probe is at least 5°C. lower than the initial, concentration-adjusted melting temperature ofsaid limiting primer.
 115. The kit according to claim 114 wherein theinitial, concentration-adjusted melting temperature of said probe is atleast 10° C. lower than the initial, concentration-adjusted meltingtemperature of said limiting primer.
 116. The kit according to claim 110further comprising directions specifying a detection step followingprimer annealing for at least some PCR cycles and specifying anannealing temperature or temperatures to be used for annealing thelimiting primer, wherein the initial, concentration-adjusted meltingtemperature of said probe is at least 5° C. below the mean of saidannealing temperature or temperatures.
 117. The kit according to claim116, wherein the initial, concentration-adjusted melting temperature ofsaid probe is at least 10° C. below the mean of said annealingtemperature or temperatures.
 118. The kit according to claim 116,wherein the melting temperature of said amplicon exceeds the initial,concentration-adjusted melting temperature of the excess primer by notmore than 18° C.
 119. The kit according to claim 118, wherein theconcentration-adjusted melting temperature of said amplicon exceeds theinitial, concentration-adjusted melting temperature of the excess primerby 7-15° C.
 120. A kit of reagents for performing a homogenouspolymerase chain reaction (PCR) assay for at least one pre-selected DNAtarget sequence comprising a thermostable DNA polymerase, dNTP's and,for each target sequence, a pair of matched PCR limiting primers forproducing a first amplicon, an excess primer for producing a second,single-stranded amplicon utilizing said first amplicon as a template,and a labeled hybridization probe that emits a signal indicative of thepresence of said second amplicon, wherein said limiting primers are tobe used in a first, equimolar, amount at a concentration of up to 200nM, said excess primer is to be used in a second amount, at least fivetimes greater than said first amount, and at a concentration at leastfive times greater than the concentration of the limiting primers, andwherein the initial, concentration-adjusted melting temperature of theexcess primer is at least 5° C. lower than the ii initial,concentration-adjusted melting temperatures of the limiting primers.121. The kit according to claim 120 wherein the melting temperature ofthe second amplicon exceeds the initial, concentration-adjusted meltingtemperature of the excess primer by not more than 25° C.
 122. The kitaccording to claim 120 wherein said second amount is at least ten timesgreater than said first amount and wherein said excess primer is to beused at a concentration at least ten times greater than the limitingprimers concentration.